|
2006 FCS papers |
Adjimatera, N., T. Kral, M. Hof and
I. S. Blagbrough. (2006) Lipopolyamine-Mediated Single Nanoparticle
Formation of Calf Thymus DNA Analyzed by Fluorescence Correlation Spectroscopy.
Pharmaceutical Research 23(7):1564-1573.
The
aim of this study is to analyze linear calf thymus DNA (ct DNA) nanoparticle
formation with N4,N9-dioleoylspermine and N1-cholesteryl spermine carbamate.
Fluorescence correlation spectroscopy (FCS) was used to det. the quality of ct
DNA condensed by lipopolyamines. ct DNA was prelabeled with PicoGreen (PG) to
allow fluorescence intensity fluctuation measurement and anal.
N4,N9-Dioleoylspermine efficiently condensed ct DNA into point-like mols. with
diffusion coeff. (D) = 1.8 * 10-12 m2/s and particle no. (PN) = 0.7 [at ammonium/phosphate
(N/P) charge ratio=1.0-1.5]. The detd. PN values are close to the theor. value
of 0.6, providing evidence that the DNA conformation has been fully
transformed, and thus a single nanoparticle has been detected. N1-Cholesteryl
spermine carbamate showed (slightly) poorer DNA condensation efficiency, even
at higher N/P ratios (N/P = 1.5-2.5) with D = 1.3 * 10-12 m2/s and PN value of
5.2. N4,N9-Dioleoylspermine is a more efficient DNA-condensing agent than
N1-cholesteryl spermine carbamate. FCS measurement using PG as the probe is a
novel anal. method to detect single nanoparticles of condensed DNA in nonviral
gene therapy formulation studies.
Allen, N. W. and N. L. Thompson.
(2006) Ligand binding by estrogen receptor beta attached to nanospheres
measured by fluorescence correlation spectroscopy. Cytometry, Part A 69A(6):524-532.
Although
many indirect methods have been chosen to study the system of estrogen receptor
ligand binding, an ideal method is fluorescence correlation spectroscopy (FCS).
FCS is nondestructive to the sample, uses very small sample vols., and operates
well within physiol. concn. ranges. The methodol. was developed to biotinylate
the estrogen receptor b-ligand binding domain (ERb-LBD) using biotin with a
very short spacer and to then attach this protein to a 40 nm neutravidin-coated
bead (nanosphere). Diffusional FCS data were obtained for a fluorescently
labeled coactivator peptide, steroid receptor coactivator peptide-1
(A-SRC-1(2)), in the absence and presence of bead-bound ERb-LBD. Data were also
acquired in the presence of one of the endogenous ligands for ERb,
17b-estradiol, and with tamoxifen. The bead strategy resulted in a decreased
receptor diffusion coeff. and consequent increase in the decay time of the FCS
autocorrelation functions for receptor-bound, labeled SRC-1(2). Thus, free and
bound coactivators were much more readily distinguished by FCS. Discrimination
between the fluorescently labeled unbound and bound species could be detd. in
autocorrelation functions obtained in as few as 30 s. The advantage of using
FCS with the ERb-LBD: bead methodol. is the ability to obtain reliable and
reproducible data in a short time frame.
Ambjornsson, T., S. K. Banik, O.
Krichevsky and R. Metzler. (2006) Sequence Sensitivity of Breathing Dynamics
in Heteropolymer DNA. Physical Review Letters 97(12):128105/1-128105/4.
The
authors study the fluctuation dynamics of localized denaturation bubbles in
heteropolymer DNA with a master equation and complementary stochastic
simulation based on novel DNA stability data. A significant dependence of
opening probability and waiting time between bubble events on the local DNA
sequence is revealed and quantified for a biol. sequence of the T7
bacteriophage. Quant. agreement with data from fluorescence correlation
spectroscopy is demonstrated.
Andrews, A. B., R. E. Guerra, O. C.
Mullins and P. N. Sen. (2006) Diffusivity of Asphaltene Molecules by
Fluorescence Correlation Spectroscopy. Journal of Physical Chemistry A 110(26):8093-8097.
Using
fluorescence correlation spectroscopy (FCS) the authors measure the
translational diffusion coeff. of asphaltene mols. in toluene at extremely low
concns. (0.03-3.0 mg/L): where aggregation does not occur. The translational
diffusion coeff. of asphaltene mols. in toluene is .apprx.0.35 * 10-5 cm2/s at
room temp. This diffusion coeff. corresponds to a hydrodynamic radius of
.apprx.1 nm. These data confirm previously estd. size from rotational diffusion
studied using fluorescence depolarization. The implication of this concurrence
is that asphaltene mol. structures are monomeric, not polymeric.
Aujard, I., C. Benbrahim, M. Gouget,
O. Ruel, J.-B. Baudin, P. Neveu and L. Jullien. (2006) o-Nitrobenzyl
photolabile protecting groups with red-shifted absorption: syntheses and
uncaging cross-sections for one- and two-photon excitation. Chemistry--A
European Journal 12(26):6865-6879.
The
o-nitrobenzyl platform for designing photolabile protecting groups with
red-shifted absorption that could be photolyzed upon one- and two-photon
excitation is evaluated. Several synthetic pathways to build different
conjugated o-nitrobenzyl backbones, as well as to vary the benzylic position,
are reported. Relative to the 4,5-dimethoxy-2-nitrobenzyl group, several
o-nitrobenzyl derivs. exhibit a large and red-shifted one-photon absorption
within the near-UV range. Uncaging after one-photon excitation was studied by
measuring UV-visible absorption and steady-state fluorescence emission on model
caged ethers and esters. In the whole series investigated, the caged substrates
were released cleanly upon photolysis. Quantum yields of uncaging after
one-photon absorption lie within the 0.1-1% range. The quantum yields decrease
when the max. wavelength absorption of the o-nitrobenzyl protecting group is
increased. A new method based on fluorescence correlation spectroscopy (FCS)
after two-photon excitation was used to measure the action uncaging cross
section for two-photon excitation. The series of o-nitrobenzyl caged
fluorescent coumarins investigated exhibit values within the 0.1-0.01
Goeppert-Mayer (GM) range. Such results are in line with the low quantum yields
of uncaging assocd. with cross-sections of 1-50 GM for two-photon absorption.
Although the cross-sections for one- and two-photon absorption of o-nitrobenzyl
photolabile protecting groups can be readily improved, the difficulty in
enlarging the corresponding action uncaging cross-sections in view of the obsd.
trend of their quantum yield of uncaging should be emphasized.
Barker, J., S. Courtney, T.
Hesterkamp, D. Ullmann and M. Whittaker. (2006) Fragment screening by
biochemical assay. Expert Opinion on Drug Discovery 1(3):225-236.
A
review. The use of high concn. biochem. assays to identify weak binding
fragment mols. can be an effective method to identify novel starting points for
medicinal chem. programs. The combination of a high-quality fragment library
with sensitive biochem. screening methods is a viable alternative to the more
commonly used fragment screening methods such as NMR screening or
high-throughput X-ray crystallog. Notably, there are a no. of literature
reports where fragment mols. have been identified by a high concn. biochem.
assay. The use of high concn. screening of fragments using a portfolio of
single-mol. fluorescence correlation spectroscopy detection techniques to
ensure the highest reproducibility and sensitivity have been demonstrated, as
well as the use of and X-ray crystallog. to det. the binding mode of active
fragments.
Bates, I. R., B. Hebert, Y. Luo, J.
Liao, A. I. Bachir, D. L. Kolin, P. W. Wiseman and J. W. Hanrahan. (2006) Membrane
lateral diffusion and capture of CFTR within transient confinement zones.
Biophysical Journal 91(3):1046-1058.
The
cystic fibrosis transmembrane conductance regulator (CFTR) channel interacts
with scaffolding and other proteins that are expected to restrict its lateral
movement, yet previous studies have reported predominantly free diffusion. We
examd. the lateral mobility of CFTR channels in live baby hamster kidney cells
using three complementary methods. Channels bearing an extracellular
biotinylation target sequence were labeled with streptavidin conjugated with
fluorescent dyes (Alexa Fluor 488 or 568) or quantum dots (qDot605).
Fluorescence recovery after photobleaching and image correlation spectroscopy
of the dye-labeled channels revealed a significant immobile population
(.apprx.50%), which was confirmed by direct single particle tracking (SPT) of
qDot605-labeled CFTR. Adding 10 histidine residues at the C-terminus of CFTR to
mask the postsynaptic d. 95, Disks large, ZO-1 (PDZ)-binding motif abolished
its assocn. with EBP50/NHERF1, reduced the immobile fraction, and increased
mobility. Other interactions that are not normally detected on this timescale
became apparent when binding of PDZ domain proteins was disrupted. SPT revealed
that CFTRHis-10 channels diffuse randomly, become immobilized for periods
lasting up to 1 min, and in some instances are recaptured at the same location.
The impact of transient confinement on the measured diffusion using the three
fluorescence techniques were assessed using computer simulations of the biol.
expts. Finally, the impact of endosomal CFTR on mobility measurements was
assessed by fluorescence correlation spectroscopy. These results reveal
unexpected features of CFTR dynamics which may influence its ion channel
activity.
Bates, I. R., P. W. Wiseman and J.
W. Hanrahan. (2006) Investigating membrane protein dynamics in living cells.
Biochemistry and Cell Biology 84(6):825-831.
A
review. Live cell imaging is a powerful tool for understanding the function and
regulation of membrane proteins. In this review, we briefly discuss 4 fluorescence-microscopy-based
techniques for studying the transport dynamics of membrane proteins:
fluorescence-correlation spectroscopy, image-correlation spectroscopy,
fluorescence recovery after photobleaching, and single-particle and (or) mol.
tracking. The advantages and limitations of each approach are illustrated using
recent studies of an ion channel and cell adhesion mols.
Bayer, J. and J. O. Raedler. (2006) DNA
microelectrophoresis using double focus fluorescence correlation spectroscopy.
Electrophoresis 27(20):3952-3963.
Double
focus fluorescence correlation spectroscopy (dfFCS) was used to det.
electrophoretic mobilities of short double-stranded DNA (dsDNA)-fragments (75
base pairs (bp) -1019 bp) in microfluidic channels. The electrokinetic flow
profile across a microchannel was measured with 1 mm spatial resoln. and sepd.
in electroosmotic and electrophoretic contributions. Expts. show that the free
soln. mobility is independent of DNA length. The diffusion const. is addnl.
detd. by FCS and follows a length dependent rod-diffusion model. We interpret
the electrophoretic mobilities using a modified Nernst Einstein relation, which
addnl. takes Manning condensation and counterion induced hydrodynamic
retardation forces into account. In 3% w/v polyethylene oxide (PEO)-network (Mr
3 . 105 Dalton) the electrophoretic velocities become size-dependent with a
power-law exponent between 0.28 and 0.31. Mixts. of dsDNA-fragments exhibit
distinguishable peaks in the dfFCS cross-correlation function. The potential of
dfFCS for realtime micro-anal. in terms of speed and spatial resoln. is
discussed.
Becker, W. and A. Bergmann. (2006) Multidimensional
time-correlated single photon counting. Proceedings of SPIE-The
International Society for Optical Engineering 6372(Advanced Photon
Counting Techniques):637201/1-637201/10.
Time-correlated
single photon counting (TCSPC) is based on the detection of single photons of a
periodic light signal, measurement of the detection time of the photons, and
the build-up of the photon distribution vs. the time in the signal period.
TCSPC achieves a near ideal counting efficiency and transit-time-spread-limited
time resoln. for a given detector. The drawback of traditional TCSPC is the low
count rate, long acquisition time, and the fact that the technique is
one-dimensional, i.e. limited to the recording of the pulse shape of light
signals. We present an advanced TCSPC technique featuring multi-dimensional
photon acquisition and a count rate close to the capability of currently available
detectors. The technique is able to acquire photon distributions vs.
wavelength, spatial coordinates, and the time on the ps scale, and to record
fast changes in the fluorescence lifetime and fluorescence intensity of a
sample. Biomedical applications of advanced TCSPC techniques are time-domain
optical tomog., recording of transient phenomena in biol. systems, spectrally
resolved fluorescence lifetime imaging, FRET expts. in living cells, and the
investigation of dye-protein complexes by fluorescence correlation
spectroscopy. We demonstrate the potential of the technique for selected
applications.
Bernheim-Groswasser, A., R.
Shusterman and O. Krichevsky. (2006) Fluorescence correlation spectroscopy
analysis of segmental dynamics in actin filaments. Journal of Chemical
Physics 125(8):084903/1-084903/11.
We
adapt fluorescence correlation spectroscopy (FCS) formalism to the studies of
the dynamics of semiflexible polymers and derive expressions relating FCS
correlation function to the longitudinal and transverse mean-square
displacements (MSDs) of polymer segments. The obtained relations do not depend
on any specific model of polymer dynamics. We use the derived expressions to
measure the dynamics of actin filaments in two exptl. situations: filaments
labeled at distinct positions and homogeneously labeled filaments. Both
approaches give consistent results and allow measurement of the temporal
dependence of the segmental mean-square displacement over almost five decades
in time, from .apprx.40 ms to .apprx.2 s. These noninvasive measurements allow
for a detailed quant. comparison of the exptl. data to the current theories of
semiflexible polymer dynamics. Good quant. agreement is found between the
exptl. results and theories explicitly accounting for the hydrodynamic
interactions between polymer segments.
Bi, R., P. D. Zhang, C. Q. Dong and
J. C. Ren. (2006) Combination of micro-fluidic chip with fluorescence
correlation spectroscopy for single molecule detection. Chinese Chemical
Letters 17(4):521-524.
A
single mol. detection technique was developed by the combination of a single
channel poly(dimethylsiloxane)/glass micro-fluidic chip and fluorescence
correlation spectroscopy (FCS). This method was successfully used to det. the
proportion of two model components in the mixt. contg. fluorescein and
rhodamine-green succinimidyl ester.
Birkmann, E., O. Schaefer, N.
Weinmann, C. Dumpitak, M. Beekes, R. Jackman, L. Thorne and D. Riesner. (2006) Detection
of prion particles in samples of BSE and scrapie by fluorescence correlation
spectroscopy without proteinase K digestion. Biological Chemistry 387(1):95-102.
A
characteristic feature of prion diseases such as bovine spongiform
encephalopathy (BSE) is the accumulation of a pathol. isoform of the host-encoded
prion protein, PrP. In contrast to its cellular isoform PrPc, the pathol.
isoform PrPSc forms insol. aggregates. All com. BSE tests currently used for
routine testing are based on the proteinase K (PK) resistance of PrP, but not
all pathol. PrP is PK-resistant. In the present study, single prion particles
were counted by fluorescence correlation spectroscopy (FCS). The property of PK
resistance is not required, i.e., both the PK-resistant and the PK-sensitive
parts of the prion particles are detectable. PrP aggregates were prepd. from
the brains of BSE-infected cattle, as well as from scrapie-infected hamsters,
by the NaPTA pptn. method without PK digestion. They were labeled using 2
different PrP-specific antibodies for FCS measurements in the dual-color mode
(2D-FIDA). Within the limited no. of samples tested, BSE-infected cattle and
scrapie-infected hamsters in the clin. stage of the disease could be
distinguished with 100% specificity from a control group. Thus, a diagnostic
tool for BSE detection with complete avoidance of PK treatment is presented,
which should have particular advantages for testing animals in the preclin.
stage.
Blancquaert, Y., A. Delon, J.
Derouard and R. Jaffiol. (2006) Spatial fluorescence cross-correlation
spectroscopy between core and ring pinholes. Proceedings of SPIE-The
International Society for Optical Engineering 6191(Biophotonics and New
Therapy Frontiers):61910B/1-61910B/9.
Fluorescence
Correlation Spectroscopy (FCS) is an attractive method to measure mol. concn.,
mobility parameters and chem. kinetics. However its ability to discriminate
different diffusing species needs to be improved. Recently, the authors have
proposed a simplified spatial Fluorescence cross Correlation Spectroscopy
(sFCCS) method, allowing, with only one focused laser beam to obtain two
confocal vols. spatially shifted. Now, the authors present a new sFCCS optical
geometry where the two pinholes, a ring and core, are encapsulated one in the
other. In this approach all phys. and chem. processes that occur in a single
vol., like singlet-triplet dynamics and photobleaching, can be eliminated;
moreover, this new optical geometry optimizes the collection of fluorescence.
The first cross Correlation curves for Rhodamine 6G (Rh6G) in Ethanol are
presented, in addn. to the effect of the size of fluorescent particules
(nano-beads, diams. : 20, 100 and 200 nm). The relative simplicity of the
method leads the authors to propose sFCCS as an appropriate method for the
detn. of diffusion parameters of fluorophores in soln. or cells. Nevertheless,
progresses in the engineering of the optical Mol. Detection Efficiency vols.
are highly desirable, to improve the discrimination between the cross
correlated vols.
Bosco, S. J., H. Zettl, J. J.
Crassous, M. Ballauff and G. Krausch. (2006) Interactions between Methyl
Cellulose and Sodium Dodecyl Sulfate in Aqueous Solution Studied by Single
Molecule Fluorescence Correlation Spectroscopy. Macromolecules 39(25):8793-8798.
The
interactions between the anionic surfactant sodium dodecyl sulfate (SDS) and a
hydrophobically modified nonionic polymer, Me cellulose (MC), have been
investigated in aq. soln. by fluorescence correlation spectroscopy (FCS) and
rheol. FCS is used to follow the dynamics of different populations of single
aggregates. We are able to follow the soln. properties over a wide concn. range
of both polymer and surfactant. At const. MC concn. the diffusion time of
single aggregates increases gradually up to a certain SDS concn. and decreases
to a min. when the SDS concn. is further increased. This behavior coincides
with the behavior of the zero shear viscosity. A model is proposed to explain
the effect of surfactant concn. on polymer conformation and aggregation size.
Braenden, M., T. Sanden, P.
Brzezinski and J. Widengren. (2006) Localized proton microcircuits at the
biological membrane-water interface. Proceedings of the National Academy of
Sciences of the United States of America 103(52):19766-19770.
Cellular
processes such as nerve conduction, energy metab., and import of nutrients into
cells all depend on transport of ions across biol. membranes through
specialized membrane-spanning proteins. Understanding these processes at a mol.
level requires mechanistic insights into the interaction between these proteins
and the membrane itself. To explore the role of the membrane in ion
translocation we used an approach based on fluorescence correlation
spectroscopy. Specifically, we investigated exchange of protons between the
water phase and the membrane surface, as well as diffusion of protons along
membrane surfaces, at a single-mol. level. We show that the lipid head groups
collectively act as a proton-collecting antenna, dramatically accelerating
proton uptake from water to a membrane-anchored proton acceptor. Furthermore,
the results show that proton transfer along the surface can be significantly
faster than that between the lipid head groups and the surrounding water phase.
Thus, ion translocation across membranes and between the different membrane
protein components is a complex interplay between the proteins and the membrane
itself, where the membrane acts as a proton-conducting link between
membrane-spanning proton transporters.
Breuer, S., H. Gerlach, B. Kolaric,
C. Urbanke, N. Opitz and M. Geyer. (2006) Biochemical Indication for
Myristoylation-Dependent Conformational Changes in HIV-1 Nef. Biochemistry 45(7):2339-2349.
The
accessory HIV-1 Nef protein is essential for viral replication, high virus
load, and progression to AIDS. These functions are mediated by the alteration
of signaling and trafficking pathways and require the membrane assocn. of Nef
by its N-terminal myristoylation. However, a large portion of Nef is also found
in the cytosol, in line with the observation that myristoylation is only a weak
lipidation anchor for membrane attachment. We performed biochem. studies to
analyze the implications of myristoylation on the conformation of Nef in aq.
soln. To establish an in vivo myristoylation assay, we first optimized the
codon usage of Nef for Escherichia coli expression, which resulted in a 15-fold
higher protein yield. Myristoylation was achieved by coexpression with the
N-myristoyltransferase and confirmed by mass spectrometry. The myristoylated
protein was sol., and proton NMR spectra confirmed proper folding. Size
exclusion chromatog. revealed that myristoylated Nef appeared of smaller size
than the unmodified form but not as small as an N-terminally truncated from of
Nef that omits the anchor domain. Western blot stainings and limited proteolysis
of both forms showed different recognition profiles and degrdn. pattern. Anal.
ultracentrifugation revealed that myristoylated Nef prevails in a monomeric
state while the unmodified form exists in an oligomeric equil. of monomer,
dimer, and trimer assocns. Finally, fluorescence correlation spectroscopy using
multiphoton excitation revealed a shorter diffusion time for the lipidated
protein compared to the unmodified form. Taken together, our data indicated
myristoylation-dependent conformational changes in Nef, suggesting a rather
compact and monomeric form for the lipidated protein in soln.
Chen, C.-S., J. Yao and R. A. Durst.
(2006) Liposome encapsulation of fluorescent nanoparticles: quantum dots and
silica nanoparticles. Journal of Nanoparticle Research 8(6):1033-1038.
Quantum
dots (QDs) and silica nanoparticles (SNs) are relatively new classes of
fluorescent probes that overcome the limitations encountered by org.
fluorophores in bioassay and biol. imaging applications. We encapsulated QDs and
SNs in liposomes and sepd. nanoparticle-loaded liposomes from unencapsulated
nanoparticles by size exclusion chromatog. Fluorescence correlation
spectroscopy was used to measure the av. no. of nanoparticles inside each
liposome. Results indicated that nanoparticle-loaded liposomes were formed and
sepd. from unencapsulated nanoparticles by using a Sepharose gel. As expected,
fluorescence self-quenching of nanoparticles inside liposomes was not obsd.
Each liposome encapsulated an av. of three QDs. These studies demonstrated that
nanoparticles could be successfully encapsulated into liposomes and provided a
methodol. to quantify the no. of nanoparticles inside each liposome by
fluorescence correlation spectroscopy.
Chen, Y., B. C. Lagerholm, B. Yang
and K. Jacobson. (2006) Methods to measure the lateral diffusion of membrane
lipids and proteins. Methods (San Diego, CA, United States) 39(2):147-153.
In
this chapter, we discuss methods to measure lateral mobility of membrane lipids
and proteins using techniques based on the light microscope. These methods
typically sample lateral mobility in very small, micron-sized regions of the
membrane so that they can be used to measure diffusion in regions of single
cells. The methods are based on fluorescence from the mols. of interest or from
light scattered from particles attached to single or small groups of membrane
lipids or proteins. Fluorescence recovery after photobleaching (FRAP),
fluorescence correlation spectroscopy (FCS) and Single particle tracking (SPT) are
presented in that order. FRAP and FCS methodologies are described for a
dedicated wide field microscope although many confocal microscopes now have
software permitting these measurement to be made; nevertheless, the principles
of the measurement are the same for a wide field or confocal microscope. SPT
can be applied to trace the movements of single fluorescent mols. in membranes
but this aspect will not be treated in detail.
Chiantia, S., N. Kahya, J. Ries and
P. Schwille. (2006) Effects of ceramide on liquid-ordered domains
investigated by simultaneous AFM and FCS. Biophysical Journal 90(12):4500-4508.
The
sphingolipid ceramides are known to influence lipid lateral organization in biol.
membranes. In particular, ceramide-induced alterations of microdomains can be
involved in several cell functions, ranging from apoptosis to immune response.
We used a combined approach of at. force microscopy, fluorescence correlation
spectroscopy, and confocal fluorescence imaging to investigate the effects of
ceramides in model membranes of biol. relevance. Our results show that physiol.
quantities of ceramide in sphingomyelin/dioleoylphosphatidylcholine/cholesterol
supported bilayers lead to a significant rearrangement of lipid lateral
organization. Our exptl. setup allowed a simultaneous characterization of both
structural and dynamic modification of membrane microdomains, induced by the
presence of ceramide. Formation of similar ceramide-enriched domains and, more
general, alterations of lipid-lipid interactions can be of crucial importance
for the biol. function of cell membranes.
Chiantia, S., J. Ries, N. Kahya and
P. Schwille. (2006) Combined AFM and two-focus SFCS study of raft-exhibiting
model membranes. ChemPhysChem 7(11):2409-2418.
Dioleoylphosphatidylcholine/sphingomyelin/cholesterol
(DOPC/SM/cholesterol) model membranes exhibit liq.-liq. phase sepn. and
therefore provide a phys. model for the putative liq.-ordered domains present
in cells. Here the authors present a combination of at. force microscopy (AFM)
imaging, force measurements, confocal fluorescence imaging and two-focus
scanning fluorescence correlation spectroscopy (two-focus SFCS) to obtain
structural and dynamical information about this model membrane system.
Partition coeffs. and diffusion coeffs. in the different phases were measured
with two-focus SFCS for numerous fluorescent lipid analogs and proteins, while
being directly related to the lateral organization of the membrane and its
mech. properties probed by AFM. Moreover the combination of these different
approaches is effective in reducing artifacts resulting from the use of a
single technique.
Cotlet, M., S. Habuchi, J. E.
Whitier, J. H. Werner, F. C. De Schryver, J. Hofkens and P. M. Goodwin. (2006) Single
molecule spectroscopic characterization of a far-red fluorescent protein
(HcRed) from the Anthozoa coral Heteractis crispa. Proceedings of SPIE-The
International Society for Optical Engineering 6098:609804/1-609804/11.
We
report on the photophys. properties of a far-red intrinsic fluorescent protein
by means of single mol. and ensemble spectroscopic methods. The green
fluorescent protein (GFP) from Aequorea victoria is a popular fluorescent
marker with genetically encoded fluorescence and which can be fused to any
biol. structure without affecting its function. GFP and its variants provide
emission colors from blue to yellowish green. Red intrinsic fluorescent
proteins from Anthozoa species represent a recent addn. to the emission color
palette provided by GFPs. Red intrinsic fluorescent markers are on high demand
in protein-protein interaction studies based on fluorescence-resonance energy
transfer or in multicolor tracking studies or in cellular investigations where
autofluorescence possesses a problem. Here we address the photophys. properties
of a far-red fluorescent protein (HcRed), a mutant engineered from a
chromoprotein cloned from the sea anemone Heteractis crispa, by using a
combination of ensemble and single mol. spectroscopic methods. We show evidence
for the presence of HcRed protein as an oligomer and for incomplete maturation
of its chromophore. Incomplete maturation results in the presence of an
immature (yellow) species absorbing/fluorescing at 490/530-nm. This yellow
chromophore is involved in a fast resonance-energy transfer with the mature
(purple) chromophore. The mature chromophore of HcRed is found to adopt two
conformations, a Transoriented form absorbing and 565-nm and non-fluorescent in
soln. and a Cis-oriented form absorbing at 590-nm and emitting at 645-nm. These
two forms co-exist in soln. in thermal equil. Excitation-power dependence
fluorescence correlation spectroscopy of HcRed shows evidence for
singlet-triplet transitions in the microseconds time scale and for cis-trans
isomerization occurring in a time scale of tens of microseconds. Single mol.
fluorescence data recorded from immobilized HcRed proteins, all point to the
presence of two classes of mols.: proteins with Cis and Trans-oriented
chromophores. Immobilization of HcRed in water-filled pores of polyvinyl alc.
leads to a polymer matrix - protein barrel interaction which results in a
'freezing' of the chromophore in a stable conformation for which non-radiative
deactivation pathways are either suppressed or reduced. As a result, proteins
with both Cis- and Trans-oriented chromophores can be detected at the single
mol. level. Polymer chain motion is suggested as a mediator for an eventual
cis-trans isomerization of the chromophore in the case of single immobilized
proteins.
Crick, S. L., M. Jayaraman, C.
Frieden, R. Wetzel and R. V. Pappu. (2006) Fluorescence correlation
spectroscopy shows that monomeric polyglutamine molecules form collapsed
structures in aqueous solutions. Proceedings of the National Academy of
Sciences of the United States of America 103(45):16764-16769.
We
have used fluorescence correlation spectroscopy measurements to quantify the
hydrodynamic sizes of monomeric polyglutamine as a function of chain length (N)
by measuring the scaling of translational diffusion times (tD) for the peptide
series (Gly)-(Gln)N-Cys-Lys2 in aq. soln. We find that tD scales with N as toNn
and therefore In(tD) = In(to) + nIn(N). The values for n and In(to) are
0.32+-0.02 and 3.04+-0.08, resp. Based on these observations, we conclude that
water is a polymeric poor solvent for polyglutamine. Previous studies have
shown that monomeric polyglutamine is intrinsically disordered. These
observations combined with our fluorescence correlation spectroscopy data
suggest that the ensemble for monomeric polyglutamine is made up of a
heterogeneous collection of collapsed structures. This result is striking
because the preference for collapsed structures arises despite the absence of
residues deemed to be hydrophobic in the sequence constructs studied. Working
under the assumption that the driving forces for collapse are similar to those
for aggregation, we discuss the implications of our results for the thermodn.
and kinetics of polyglutamine aggregation, a process that has been implicated
in the mol. mechanism of Huntington's disease. chain collapse I poor solvent.
Davis, L. M. and G. Shen. (2006) Accounting
for triplet and saturation effects in FCS measurements. Current
Pharmaceutical Biotechnology 7(4):287-301.
Fluorescence
correlation spectroscopy (FCS) is an increasingly important tool for detg. low
concns. and dynamics of mols. in soln. Oftentimes triplet transitions give rise
to fast blinking effects, which are accounted for by including an exponential
term in the fitting of the autocorrelation function (ACF). In such cases,
concomitant satn. effects also modify the amplitude and shape of the remaining
parts of the ACF. The authors review studies of triplet and satn. effects in
FCS and present a simple procedure to obtain more accurate results of particle
concns. and diffusional dynamics in expts. where triplet kinetics are evident,
or where moderate laser powers approaching satn. levels are used, for example,
to acquire sufficient photon nos. when observation times are limited. The
procedure involves use of a modified function for curve-fitting the ACF, but
there are no addnl. fitting parameters. Instead, a simple calibration of the
total fluorescence count rate as a function of relative laser power is fit to a
polynomial, and the nonlinear components of this fit, together with the
relative laser power used for the FCS measurement, are used to specify the
magnitude of addnl. terms in the fitting function. Monte Carlo simulations and
expts. using Alexa dyes and quantum dots, with continuous and pulsed laser
excitation, demonstrate the application of the modified fitting procedure with
first order correction terms, in the regime where distortions in the ACF due to
photobleaching and detector dead time are small compared to those of
fluorescence satn. and triplet photophysics.
Dedecker, P., J.-i. Hotta, R. Ando,
A. Miyawaki, Y. Engelborghs and J. Hofkens. (2006) Fast and reversible
photoswitching of the fluorescent protein Dronpa as evidence by fluorescence
correlation spectroscopy. Biophysical Journal 91(5):L45-L47.
Controlling
mol. properties through photoirradn. holds great promise for its potential for
noninvasive and selective manipulation of matter. Photochromism has been obsd.
for several different mols., including green fluorescent proteins, and recently
the discovery of a novel photoswitchable green fluorescent protein called
Dronpa was reported. Dronpa displays reversible and highly efficient on/off
photoswitching of its fluorescence emission, and reversible switching of
immobilized single mols. of Dronpa with response times faster than 20 ms was
demonstrated. In this Letter, we expand these observations to freely diffusing
mols. by using fluorescence correlation spectroscopy with simultaneous
excitation at 488 and 405 nm. By varying the intensity of irradn. at 405 nm, we
demonstrate the reversible photoswitching of Dronpa under these conditions, and
from the obtained autocorrelation functions we conclude that this
photoswitching can occur within tens of microseconds.
DeRouchey, J., G. F. Walker, E.
Wagner and J. O. Raedler. (2006) Decorated Rods: A \"Bottom-Up\"
Self-Assembly of Monomolecular DNA Complexes. Journal of Physical Chemistry
B 110(10):4548-4554.
Fluorescence
correlation spectroscopy (FCS) and gel electrophoresis measurements are
performed to investigate both the no. and size of complexes of linear
double-stranded DNA (dsDNA) fragments with 1:1 diblock copolymers consisting of
a cationic moiety, branched polyethyleneimine (bPEI) of 2, 10, or 25 kDa,
covalently bound to a neutral shielding moiety, poly(ethylene glycol) (PEG; 20
kDa). By systematically decreasing the bPEI length, the PEG grafting d. along
the DNA chain can be directly controlled. For 25 and 10 kDa bPEI-PEG copolymers,
severe aggregation is obsd. despite the presence of the shielding PEG. Upon
decreasing the bPEI length to 2 kDa, controlled self-assembly of monomol. DNA
nanoparticles is obsd. The resulting complexes are in quant. agreement with a
theor. model based on a single DNA encased in a dense PEG polymer brush layer.
The resulting PEGylated complexes show high stability against both salt and
protein and hence are of potential use for in vivo gene delivery studies.
Dertinger, T., I. Gregor, I. von der
Hocht, R. Erdmann, B. Kraemer, F. Koberling, R. Hartmann and J. Enderlein.
(2006) Measuring precise diffusion coefficients with two-focus fluorescence
correlation spectroscopy. Proceedings of SPIE-The International Society for
Optical Engineering 6092(Ultrasensitive and Single-Molecule Detection
Technologies):609203/1-609203/5.
We
present a new method for precisely measuring diffusion coeffs. of fluorescent
mols. at nanomolar concns. The method is based on a modified Fluorescence
Correlation Spectroscopy (FCS)-setup which is robust against many artifacts
that are inherent to std. FCS1, 2. The core idea of the new method is the
introduction of an external ruler by generating two laterally shifted and
overlapping laser foci at a fixed and known distance. Data fitting is
facilitated by ab initio calcns. of resulting correlation curves and subsequent
affine transformation of these curves to match the measured auto- and
cross-correlation functions. The affine transformation coeff. along the time
axis then directly yields the correct diffusion coeff. This method is not
relying on the rather inexact assumption of a 3D Gaussian shaped detection vol.
We measured the diffusion coeff. of the red fluorescent dye Atto-655 (Atto-Tec
GmbH) in water and compared the obtained value with results from Gradient
Pulsed Field NMR (GPF-NMR).
Dertinger, T., I. von Hocht, A.
Benda, M. Hof and J. Enderlein. (2006) Surface Sticking and Lateral
Diffusion of Lipids in Supported Bilayers. Langmuir 22(22):9339-9344.
The
diffusion of fluorescently labeled lipids in supported bilayers is studied
using two different methods: Z-scan fluorescence correlation spectroscopy
(z-scan FCS) and two-focus fluorescence correlation spectroscopy (2f-FCS). It
is found that the data can be fitted consistently only when taking into account
partial sticking of the labeled lipids to the supporting glass surface. A
kinetic reaction-diffusion model is developed and applied to the data. The
authors find a very slow sticking rate which, however, when neglected, leads to
strongly varying ests. of the free diffusion coeff. The study reveals a strong
sensitivity of FCS on even slight binding/unbinding kinetics of the labeled
mols., which has significance for related diffusion measurements in cellular
lipid membranes.
Dix, J. A., E. F. Y. Hom and A. S.
Verkman. (2006) Fluorescence Correlation Spectroscopy Simulations of
Photophysical Phenomena and Molecular Interactions: A Molecular Dynamics/Monte
Carlo Approach. Journal of Physical Chemistry B 110(4):1896-1906.
Fluorescence
correlation spectroscopy (FCS) is being applied increasingly to study diffusion
and interactions of fluorescently labeled macromols. in complex biol. systems.
Fluctuations in detected fluorescence, dF(t), are expressed as time-correlation
functions, G(t), and photon-count histograms, P(k;DT). Here, we developed a
generalized simulation approach to compute G(t) and P(k;DT) for complex systems
with arbitrary geometry, photophysics, diffusion, and macromol. interactions.
G(t) and P(k;DT) were computed from dF(t) generated by a Brownian dynamics
simulation of single-mol. trajectories followed by a Monte Carlo simulation of
fluorophore excitation and detection statistics. Simulations were validated by
comparing anal. and simulated G(t) and P(k;DT) for diffusion of noninteracting
fluorophores in a three-dimensional Gaussian excitation and detection vol.
Inclusion of photobleaching and triplet-state relaxation produced significant
changes in G(t) and P(k;DT). Simulations of macromol. interactions and complex
diffusion were done, including transient fluorophore binding to an immobile
matrix, cross-correlation anal. of interacting fluorophores, and anomalous sub-
and superdiffusion. The computational method developed here is generally
applicable for simulating FCS measurements on systems complicated by
fluorophore interactions or mol. crowding, and exptl. protocols for which G(t)
and P(k;DT) cannot be computed anal.
Dong, C., R. Bi, H. Qian, L. Li and
J. Ren. (2006) Coupling fluorescence correlation spectroscopy with microchip
electrophoresis to determine the effective surface charge of water-soluble
quantum dots. Small 2(4):534-538.
A
new method to det. the surface charge of quantum dots by coupling fluorescence
correlation spectroscopy with microchip electrophoresis was investigated. The
technique was used to det. the surface charge of different stabilizer-modified
CdTe QDs and to study their transport properties in elec. fields. The surface
charge of QDs was closely assocd. with the type of stabilizer on the QD
surface, the buffer pH value, and other factors.
Dong, C., H. Qian, N. Fang and J.
Ren. (2006) Study of Fluorescence Quenching and Dialysis Process of CdTe
Quantum Dots, Using Ensemble Techniques and Fluorescence Correlation
Spectroscopy. Journal of Physical Chemistry B 110(23):11069-11075.
Luminescence
properties of quantum dots (QDs) are closely related to their surface structure
and chem. properties. In this work some ensemble techniques and fluorescence
correlation spectroscopy (FCS) were used to study the fluorescence quenching
and dialysis process of CdTe QDs. It is found that when some heavy metal ions,
such as silver ions (Ag+), quench QDs, the free Ag+ ions bind with bare Te
atoms and form the AgTe structure on the surface. The FCS exptl. results show
that the quenching process is not the gradual redn. of fluorescence intensity
of single QDs, but the decrease in the no. of bright QDs with the addn. of Ag+
ions. In other words, the bright QDs turn into dark directly in the quenching
process. It is obsd. that some dark QDs converse into the bright QDs in the
dialysis expts. and the dialysis process can improve the brightness per QDs.
Furthermore, the results of FCS and fluorescence spectroscopy illustrate that
the increase of the fluorescence quantum yield (QY) is mainly attributed to the
removal of excess unreacted Cd-MPA complex and the possible chem. change of the
QDs surface in the dialysis process. These new results can help us to further
understand the complex surface structure of water-sol. QDs, improve their
surface chem. features, and expand their applications in some fields.
Donsmark, J., L. Jorgensen, S.
Mollmann, S. Frokjaer and C. Rischel. (2006) Kinetics of Insulin Adsorption
at the Oil-Water Interface and Diffusion Properties of Adsorbed Layers
Monitored Using Fluorescence Correlation Spectroscopy. Pharmaceutical
Research 23(1):148-155.
The
adsorption of insulin at an oil-water interface was studied with fluorescence
correlation spectroscopy (FCS). FCS is able to measure diffusion properties of
insulin at nanomolar concns., making it possible to detect the very early steps
in the adsorption process. Below 20 nM bulk insulin concn., the insulin mols.
adsorbed to the surface diffuse freely at all times during the expt. (a few
hours). At higher concns., a surprisingly abrupt transition to a slow diffusion
phase is obsd. Based on the information about both diffusion times and mol.
brightness derived from the FCS expts., the authors suggest that the transition
represents the formation of a fractal network. FCS may be a valuable tool in
pharmaceutical formulation science, because it provides information about
concn. buildup and phase changes at interfaces formed in drug delivery systems.
Duval, J. F. L., V. I. Slaveykova,
M. Hosse, J. Buffle and K. J. Wilkinson. (2006) Electrohydrodynamic
Properties of Succinoglycan as Probed by Fluorescence Correlation Spectroscopy,
Potentiometric Titration and Capillary Electrophoresis. Biomacromolecules 7(10):2818-2826.
The
electrostatic, hydrodynamic and conformational properties of aq. solns. of
succinoglycan have been analyzed by fluorescence correlation spectroscopy
(FCS), proton titrn., and capillary electrophoresis (CE) over a large range of
pH values and electrolyte (NaCl) concns. Using the theor. formalism developed
previously for the electrokinetic properties of soft, permeable particles, a
quant. anal. for the electro-hydrodynamics of succinoglycan is performed by
taking into account, in a self-consistent manner, the measured values of the diffusion
coeffs., elec. charge densities, and electrophoretic mobilities. For that
purpose, two limiting conformations for the polysaccharide in soln. are tested,
i.e., succinoglycan behaves as (i) a spherical, random coil polymer or (ii) a
rodlike particle with charged lateral chains. The results show that
satisfactory modeling of the titrn. data for ionic strengths larger than 50 mM
can be accomplished using both geometries over the entire range of pH values.
Electrophoretic mobilities measured for sufficiently large pH values (pH >
5-6) are in line with predictions based on either model. The best manner to
discriminate between these two conceptual models is briefly discussed. For low
pH values (pH < 5), both models indicate aggregation, resulting in an increase
of the hydrodynamic permeability and a decrease of the diffusion coeff.
Eggeling, C., J. Widengren, L.
Brand, J. Schaffer, S. Felekyan and C. A. M. Seidel. (2006) Analysis of
photobleaching in single-molecule multicolor excitation and Foerster resonance
energy transfer measurements. Journal of Physical Chemistry A 110(9):2979-2995.
Dye
photobleaching is a major constraint of fluorescence readout within a range of
applications. The authors studied the influence of photobleaching in fluorescence
expts. applying multicolor laser as well as Foerster resonance energy transfer
(FRET) mediated excitation using several red-emitting dyes frequently used in
multicolor expts. or as FRET acceptors. The chosen dyes (cyanine 5 (Cy5),
MR121, Alexa660, Alexa680, Atto647N, Atto655) have chem. distinct chromophore
systems and can be excited at 650 nm. Several fluorescence anal. techniques
were applied to detect photobleaching and to disclose the underlying
photophysics, all of which are based on single-mol. detection: (1) fluorescence
correlation spectroscopy (FCS) of bulk solns., (2) fluorescence
cross-correlation of single-mol. trajectories, and (3) multiparameter
fluorescence detection (MFD) of single-mol. events. The max. achievable
fluorescence signals as well as the survival times of the red dyes were
markedly reduced under addnl. laser irradn. in the range of 500 nm.
Particularly at excitation levels at or close to satn., the 500 nm irradn.
effectively induced transitions to higher excited electronic states on already
excited dye mols., leading to a pronounced bleaching reactivity. A theor. model
for the obsd. laser irradiance dependence of the fluorescence brightness of a
Cy5 FRET acceptor dye was developed introducing the full description of the
underlying photophysics. The model takes into account acceptor as well as donor
photobleaching from higher excited electronic states, population of triplet
states, and energy transfer to both the ground and excited states of the
acceptor dye. Also, photoinduced reverse intersystem crossing via higher
excited triplet states is included, which is very efficient for Cy5 attached to
DNA. Comparing continuous wave (cw) and pulsed donor excitation, a strong
enhancement of acceptor photobleaching by a factor of 5 was obsd. for the
latter. Thus, in the case of fluorescence expts. using multicolor pulsed laser
excitation, the application of the appropriate timing of synchronized green and
red laser pulses in an alternating excitation mode can circumvent excessive photobleaching.
Also, important new single-mol. anal. diagnosis tools are presented: (1) For
the case of excessive acceptor photobleaching, cross-correlation anal. of
single-mol. trajectories of the fluorescence signal detected in the donor and
acceptor detection channels and vice versa shows an anticorrelated exponential
decay and growth, resp. (2) The time difference, Tg - Tr, of the mean
observation times of all photons detected for the donor and acceptor detection
channels within a single-mol. fluorescence burst allows one to identify and
exclude mols. with an event of acceptor photobleaching. The presented
single-mol. anal. methods can be constrained to, for example, FRET-active
subpopulations, reducing bias from FRET-inactive mols. The observations made are
of strong relevance for and demand a careful choice of laser action in
multicolor and FRET expts., in particular when performed at or close to satn.
Fatin-Rouge, N., K. J. Wilkinson and
J. Buffle. (2006) Combining Small Angle Neutron Scattering (SANS) and
Fluorescence Correlation Spectroscopy (FCS) Measurements To Relate Diffusion in
Agarose Gels to Structure. Journal of Physical Chemistry B 110(41):20133-20142.
Small
angle neutron scattering (SANS) and fluorescence correlation spectroscopy (FCS)
measurements were carried out on agarose hydrogels to link their microscopic
structure to the diffusivity of solutes at different scales. SANS allowed for
the detn. of the distribution of void vols. within the gels. They were shown to
be compatible with a random network of cylindrical fibers as described by the
Ogston model. FCS measured solute diffusivity in spaces similar in size to the
void vols., and thus, the results reflected the gel heterogeneity. Solute
diffusivity was predicted by modeling the gel as microscopic geometrical cells.
Variations in the diffusivity of solutes of different sizes could be predicted
from the structural parameters of the gel using theory, taking into account
obstruction by cylindrical cells and solute hydrodynamics. Prediction of the
FCS autocorrelation functions for solutes from a cell model demonstrated a lack
of sensitivity of this technique for multicomponent anal.
Foldes-Papp, Z. (2006) What it
means to measure a single molecule in a solution by fluorescence fluctuation spectroscopy.
Experimental and Molecular Pathology 80(3):209-218.
A
review. Traditional methodologies in micro- and nanofluidics measure biol.
mechanisms as an av. of a population of mols. as only their combined effect can
be detected. Fluorescence fluctuation spectroscopy methods such as fluorescence
correlation spectroscopy (FCS) and two-color fluorescence cross-correlation
spectroscopy (FCCS) are used as alternative exptl. approaches in ultrasensitive
analytics at the single-mol. level. However, what is the measurement time in
which one is able to study just one single mol. in soln. without immobilizing
it Existing theories are inadequate since they do not predict the meaningful
time as a function of the concn. of other mols. of the same kind in bulk soln.
This situation produces considerable concern, and exptl. hypotheses differ
according to which single-mol. detection methods are thought to have greater
validity. This subject is clearly at the forefront of research and should be of
great interest to exptl. medical scientists. As will be seen in this article,
it is worthwhile to obtain a correct form of the meaningful-time relationship
through theor. means. The new ideas are comprehensively presented, and this
relationship is a new concept at this time. The meaningful time for studying
just one mol. without immobilization specifies the time parameter in the
selfsame mol. likelihood estimator. Possible users for this concept are those
working in biotechnol. applications dealing with gene technol. Furthermore, the
concept is of interest for a great no. of medical, pharmaceutical and chem.
labs. It may serve as a foundation for further work in single-cell biol. It is
suspected that heterogeneities play a much larger role inside the cell than in
free soln. - a perfect opportunity for single-mol. studies and, thus, a novel
hypothesis regarding structure and dynamics of cellular networks is first
presented for the minimal neurotrophin network model.
Fu, Y., F. Ye, W. G. Sanders, M. M.
Collinson and D. A. Higgins. (2006) Single Molecule Spectroscopy Studies of
Diffusion in Mesoporous Silica Thin Films. Journal of Physical Chemistry B 110(18):9164-9170.
Single
mol. spectroscopy is applied in studies of diffusion and surface adsorption in
sol-gel-derived mesoporous SiO2 thin films. Mesoporous films are obtained by
spin casting surfactant-templated sols onto glass substrates. Small-angle x-ray
diffraction results are consistent with hexagonally ordered mesophases in
as-synthesized (i.e., surfactant-contg.) films. Upon calcination, a 30%
contraction and disordering of these structures occurs. Nile Red is used as a
fluorescent probe of both the as-synthesized and calcined films. It is loaded
into the samples at subnanomolar levels either prior to spin casting or after
calcination. Fluorescence imaging and single-point fluorescence time transients
show the dye mols. to be relatively mobile in the as-synthesized samples. But
the mols. appear entrapped at fixed locations in dry calcined films. In
calcined films rehydrated under high humidity conditions, the Nile Red mols.
again become mobile. Time transients obtained from the as-synthesized and
rehydrated samples provide clear evidence for frequent reversible adsorption of
the dye to the SiO2 surfaces. Autocorrelations of the time transients provide
quant. data on the mean diffusion coeffs. (D = 2.4 * 10-10 and 2.6 * 10-10
cm2/s) and mean desorption times (1/k = 25 and 40 s) for the as-synthesized and
rehydrated films, resp. The results prove both H2O and surfactant play important
roles in governing matrix interactions and mass transport.
Fukuma, H., K. Nakashima, Y. Ozaki
and I. Noda. (2006) Two-dimensional fluorescence correlation spectroscopy
IV: Resolution of fluorescence of tryptophan residues in alcohol dehydrogenase
and lysozyme. Spectrochimica Acta, Part A: Molecular and Biomolecular
Spectroscopy 65A(3-4):517-522.
Generalized
2-dimensional (2D) fluorescence correlation spectroscopy was used to resolve
the fluorescence spectra of 2 Trp residues in alc. dehydrogenase (I) and
lysozyme (II). In each protein, one Trp residue was buried in a hydrophobic
domain of the protein matrix and the other Trp residue was located at a
hydrophilic domain close to the protein-water interface. Fluorescence quenching
by iodide ion, a hydrophilic quencher, was employed as a perturbation to induce
the intensity change in the spectra. The Trp residue which was located at the
hydrophilic domain was effectively quenched by the quencher, whereas the Trp
residue located at the hydrophobic domain was protected from the quenching.
Therefore, the fluorescence of these 2 Trp residues have a different
sensitivity to the quenching, showing a different response to the concn. of the
quencher. Fluorescence spectra of the 2 Trp residues in I, which are heavily
overlapped in conventional 1-dimensional spectra, were successfully resolved by
the 2D correlation technique. From the asynchronous correlation map, it was
revealed that the quenching of Trp located at the hydrophobic part was brought
about after that of Trp located at the hydrophilic part. In contrast, the
fluorescence spectra of the 2 Trp residues could not be resolved after I was
denatured with guanidine-HCl. These results were consistent with the well-known
structure of I. Furthermore, it was elucidated that the present 2D anal. was
not interfered by Raman bands of the solvent, which sometimes bring difficulty
into the conventional fluorescence anal. Fluorescence spectra of the Trp
residues in II could not be resolved by the 2D correlation technique. The
differences between the 2 proteins were attributed to the fact that the Trp
residue in the hydrophobic site of II was not sufficiently protected from the
quenching.
Garai, K., M. Muralidhar and S.
Maiti. (2006) Fiber-optic fluorescence correlation spectrometer. Applied
Optics 45(28):7538-7542.
Fluorescence
correlation spectroscopy (FCS) is a sensitive technique used to probe size,
concn., flow velocity, and reaction kinetics in a dil. soln. Conventional FCS
spectrometers achieve this sensitivity at the cost of using bulky optics. We
demonstrate a technique that utilizes a single-mode optical fiber of 3.3 mm
mode field diam. to perform FCS measurements. We demonstrate that the technique
has adequate sensitivity to perform FCS measurements on fluorescent beads of 13
nm radius, and that the results agree with theor. predictions. Our method
potentially allows FCS to be extended to remote and in vivo applications.
Garai, K., P. Sengupta, B. Sahoo and
S. Maiti. (2006) Selective destabilization of soluble amyloid b oligomers by
divalent metal ions. Biochemical and Biophysical Research Communications 345(1):210-215.
Aggregation
of the amyloid b (Ab) peptide yields both fibrillar ppts. and sol. oligomers,
and is assocd. with Alzheimer's disease (AD). In vitro, Cu2+ and Zn2+ strongly
bind Ab and promote its pptn. However, less is known about their interactions
with the sol. oligomers, which are thought to be the major toxic species
responsible for AD. Using fluorescence correlation spectroscopy to resolve the
various sol. species of Ab, we show that low concns. of Cu2+ (1 mM) and Zn2+ (4
mM) selectively eliminate the oligomeric population (within .apprx.2 h), while
Mg2+ displays a similar effect at a higher concn. (60 mM). This uncovers a new
aspect of Ab-metal ion interactions, as pptn. is not substantially altered at
these low metal ion concns. Our results suggest that physiol. concns. of Cu2+
and Zn2+ can critically alter the stability of the toxic Ab oligomers and can
potentially control the course of neurodegeneration.
Gast, F. U., P. S. Dittrich, P.
Schwille, M. Weigel, M. Mertig, J. Opitz, U. Queitsch, S. Diez, B. Lincoln, F.
Wottawahet al. (2006) The microscopy cell (MicCell), a versatile
modular flowthrough system for cell biology, biomaterial research, and
nanotechnology. Microfluidics and Nanofluidics 2(1):21-36.
A
novel microfluidic perfusion system is described for high-resoln. microscopes.
Its modular design allows pre-coating of the coverslip surface with reagents,
biomols., or cells. A poly(dimethylsiloxane) (PDMS) layer is cast in a special
molding station, using masters made by photolithog. and dry etching of silicon
or by photoresist patterning on glass or silicon. This channel system can be
reused while the coverslip is exchanged between expts. As normal fluidic
connectors are used, the link to external, computer-programmable syringe pumps
is standardized and various fluidic channel networks can be used in the same
setup. The system can house hydrogel microvalves and microelectrodes close to
the imaging area to control the influx of reaction partners. A range of
applications is presented, including single-mol. anal. by fluorescence
correlation spectroscopy (FCS), manipulation of single mols. for
nanostructuring by hydrodynamic flow fields or the action of motor proteins,
generation of concn. gradients, trapping and stretching of live cells using
optical fibers precisely mounted in the PDMS layer, and the integration of
microelectrodes for actuation and sensing.
Gerard, M., Z. Debyser, L. Desender,
P. J. Kahle, J. Baert, V. Baekelandt and Y. Engelborghs. (2006) The
aggregation of alpha-synuclein is stimulated by FK506 binding proteins as shown
by fluorescence correlation spectroscopy. FASEB Journal 20(3):524-526,
10 1096/0fj 05-5126fje.
Aggregation
of a-synuclein (a-SYN) plays a key role in Parkinson's disease (PD). Here, the
authors used fluorescence correlation spectroscopy (FCS) to study a-SYN
aggregation in vitro and discovered that this process was clearly accelerated
by the addn. of FK506 binding proteins (FKBPs). This effect was obsd. both with
Escherichia coli SlyD FKBP and with human FKBP12, and was counteracted by
FK506, a specific inhibitor of FKBP. The a-SYN aggregates formed in the
presence of FKBP12 showed fibrillar morphol. The rotamase activity of FKBP
apparently accelerated the folding and subsequent aggregation of a-SYN. Since
FK506 and other non-immunosuppressive FKBP inhibitors are known to display
neuroregenerative and neuroprotective properties in disease models, the obsd.
inhibition of rotamase activity and a-SYN aggregation, may explain their mode
of action. These results open perspectives for the treatment of PD with
immunophilin ligands that inhibit a specific member of the FKBP family.
Gilbert, L., J. Toivola, O.
Valilehto, T. Saloniemi, C. Cunningham, D. White, A. R. Makela, E. Korhonen, M.
Vuento and C. Oker-Blom. (2006) Truncated forms of viral VP2 proteins fused
to EGFP assemble into fluorescent parvovirus-like particles. Journal of
Nanobiotechnology 4:No pp given.
Fluorescence
correlation spectroscopy (FCS) monitors random movements of fluorescent mols.
in soln., giving information about the no. and the size of for example
nano-particles. The canine parvovirus VP2 structural protein as well as
N-terminal deletion mutants of VP2 (-14, -23, and -40 amino acids) were fused
to the C-terminus of the enhanced green fluorescent protein (EGFP). The
proteins were produced in insect cells, purified, and analyzed by western
blotting, confocal and electron microscopy as well as FCS. The non-truncated
form, EGFP-VP2, diffused with a hydrodynamic radius of 17 nm, whereas the
fluorescent mutants truncated by 14, 23 and 40 amino acids showed hydrodynamic
radii of 7, 20 and 14 nm, resp. These results show that the non-truncated
EGFP-VP2 fusion protein and the EGFP-VP2 constructs truncated by 23 and by as
much as 40 amino acids were able to form virus-like particles (VLPs). The
fluorescent VLP, harboring VP2 truncated by 23 amino acids, showed a somewhat
larger hydrodynamic radius compared to the non-truncated EGFP-VP2. In contrast,
the construct contg. EGFP-VP2 truncated by 14 amino acids was not able to
assemble into VLP-resembling structures. Formation of capsid structures was
confirmed by confocal and electron microscopy. The no. of fluorescent fusion
protein mols. present within the different VLPs was detd. by FCS. In
conclusion, FCS provides a novel strategy to analyze virus assembly and gives
valuable structural information for strategic development of parvovirus-like
particles.
Golebiewska, U., A. Gambhir, G.
Hangyas-Mihalyne, I. Zaitseva, J. Radler and S. McLaughlin. (2006) Membrane-bound
basic peptides sequester multivalent (PIP2), but not monovalent (PS), acidic
lipids. Biophysical Journal 91(2):588-599.
Several
biol. important peripheral (e.g., myristoylated alanine-rich C kinase
substrate) and integral (e.g., the epidermal growth factor receptor) membrane
proteins contain clusters of basic residues that interact with acidic lipids in
the plasma membrane. Previous measurements demonstrate that the polyvalent
acidic lipid phosphatidylinositol 4,5-bisphosphate is bound electrostatically
(i.e., sequestered) by membrane-adsorbed basic peptides corresponding to these
clusters. We report three exptl. observations that suggest monovalent acidic
lipids are not sequestered by membrane-bound basic peptides. Binding of basic
peptides to vesicles does not decrease when the temp. is lowered below the
fluid-to-gel phase transition. The binding energy of Lys-13 to lipid vesicles increases
linearly with the fraction of monovalent acidic lipids. Binding of basic
peptides to vesicles produces no self-quenching of fluorescent monovalent
acidic lipids. One potential explanation for these results is that
membrane-bound basic peptides diffuse too rapidly for the monovalent lipids to
be sequestered. Indeed, our fluorescence correlation spectroscopy measurements
show basic peptides bound to phosphatidylcholine/phosphatidylserine membranes
have a diffusion coeff. approx. twofold higher than that of lipids, and those
bound to phosphatidylcholine/phosphatidylinositol 4,5-bisphosphate membranes
have a diffusion coeff. comparable to that of lipids.
Granick, S., L. Hong, L. Zhang, S.
Anthony and Y. Yu. (2006) Watching polymers diffuse at hard and soft
surfaces. PMSE Preprints 94:701.
One
of the outstanding challenges in understanding macromol. structure and function
revolves around what happens at surfaces, where the environment is distinctly
different from the better-understood case of macromols. in soln. This is
beginning to change with the advent of new techniques capable of characterizing
motion at the level of single mols. Using single-mol. imaging (SMI) and
fluorescence correlation spectroscopy (FCS) after two-photon excitation, this
lab. has quantified surface diffusion of various macromols. adsorbed onto hard
surfaces (glass), adsorbed onto soft surfaces (supported phospholipid
bilayers), and confined to nanometer spacings between mica sheets within a
surface forces app. A surprising dependence is found on the adsorbate's molar
mass and on its surface coverage, as well as (in the thin film situation) on
the spacing between mica sheets.
Hohner, A., J. Bayer and J. O.
Raedler. (2006) Wormlike lipid/DNA micelles in a non-polar solvent. European
Physical Journal E: Soft Matter 21(1):41-48.
The
phase behavior of DOPE/DOTAP-DNA complexes in phase-sepd. oil(dodecane)/water
mixts. was explored using Small Angle X-Ray Scattering (SAXS) and Fluorescence
Correlation Spectroscopy (FCS). Inverse micelles of DNA with cationic-lipid
coating were found in the oil phase. Varying the ratio between cationic and
neutral lipids a transition from wormlike to spherical structures is obsd. for
both long (~ 75000 bp) and short (30-1246 bp) DNA. In contrast to lipid/DNA
complexes in the water phase, there is no indication of condensed liq.-cryst.
structures in the non-polar phase. In fact, FCS measurements on short DNA
oligomers complexed with cationic lipid in alkane give clear evidence for
monomeric inverse micelles of DNA. Diln. series revealed a crit. lower concn.
of lipids and DNA for observing lipid/DNA micelles.
Horton, M. R., J. Raedler and A. P.
Gast. (2006) Phase behavior and the partitioning of caveolin-1 scaffolding
domain peptides in model lipid bilayers. Journal of Colloid and Interface
Science 304(1):67-76.
The
membrane binding and model lipid raft interaction of synthetic peptides derived
from the caveolin scaffolding domain (CSD) of the protein caveolin-1 have been
investigated. CSD peptides bind preferentially to liq.-disordered domains in
model lipid bilayers composed of cholesterol and an equimolar ratio of
dioleoylphosphatidylcholine (DOPC) and brain sphingomyelin. Three caveolin-1
peptides were studied: the scaffolding domain (residues 83-101), a water-insol.
construct contg. residues 89-101, and a water-sol. construct contg. residues
89-101. Confocal and fluorescence microscopy investigation shows that the
caveolin-1 peptides bind to the more fluid cholesterol-poor phase. The binding
of the water-sol. peptide to lipid bilayers was measured using fluorescence
correlation spectroscopy (FCS). We measured molar partition coeffs. of 104 M-1
between the sol. peptide and phase-sepd. lipid bilayers and 103 M-1 between the
sol. peptide and bilayers with a single liq. phase. Partial phase diagrams for
our phase-sepg. lipid mixt. with added caveolin-1 peptides were measured using
fluorescence microscopy. The water-sol. peptide did not change the phase
morphol. or the miscibility transition in giant unilamellar vesicles (GUVs);
however, the water-insol. and full-length CSD peptides lowered the liq.-liq.
melting temp.
Hosokawa, C., H. Yoshikawa and H.
Masuhara. (2006) Enhancement of biased diffusion of dye-doped nanoparticles
by simultaneous irradiation with resonance and nonresonance laser beams.
Japanese Journal of Applied Physics, Part 2: Letters & Express Letters 45(12-16):L453-L456.
We
propose and demonstrate the enhancement of the biased diffusion of dye-doped nanoparticles
using resonance and nonresonance laser beams. The Brownian motion of
nanoparticles in a laser focus is investigated by fluorescence correlation
spectroscopy (FCS) and the time variation in fluorescence intensity. From the
anal. of autocorrelation functions, it is demonstrated that the difference
between the transit times of nanoparticles in the focal spot with and without
resonance laser irradn. increases .apprx.7-fold by the simultaneous irradn. of
a near-IR laser. This method is applicable to the selective optical
manipulation of dye-stained nanomaterials and biomols. in soln.
Humpolickova, J., E. Gielen, A.
Benda, V. Fagulova, J. Vercammen, M. vande Ven, M. Hof, M. Ameloot and Y.
Engelborghs. (2006) Probing diffusion laws within cellular membranes by
Z-scan fluorescence correlation spectroscopy. Biophysical Journal 91(3):L23-L25.
The
plasma membrane of various mammalian cell types is heterogeneous in structure
and may contain microdomains, which can impose constraints on the lateral diffusion
of its constituents. Fluorescence correlation spectroscopy (FCS) can be used to
investigate the dynamic properties of the plasma membrane of living cells. Very
recently, L. Wawrezinieck et al. (2005) described a method to probe the nature
of the lateral microheterogeneities of the membrane by varying the beam size in
the FCS instrument. The dependence of the width of the autocorrelation function
at half-max., i.e., the diffusion time, on the transverse area of the confocal
vol. gives information on the nature of the imposed confinement. The authors
describe an alternative approach that yields essentially the same information,
and can readily be applied on com. FCS instruments by measuring the diffusion
time and the particle no. at various relative positions of the cell membrane
with respect to the waist of the laser beam, i.e., by performing a Z-scan.
Hwang, L. C., M. Gosch, T. Lasser
and T. Wohland. (2006) Simultaneous multicolor fluorescence
cross-correlation spectroscopy to detect higher order molecular interactions
using single wavelength laser excitation. Biophysical Journal 91(2):715-727.
Fluorescence
cross-correlation spectroscopy is a powerful method for the study of mol.
interactions and dynamics in soln. and even in living cells. Usually, in the
optical setup, either two laser beams have to be superimposed in their resp.
confocal vols. or two-photon excitation is used for a dual-color detection
system. It has been shown recently that fluorescence cross correlation can be
achieved with spectrally similar fluorophores using single wavelength
excitation fluorescence cross-correlation spectroscopy (SW-FCCS). In this
study, the authors show that SW-FCCS allows the simultaneous excitation of up
to three fluorophores in which the cross correlation of their fluctuation
signals is detected sep. in three detection channels. The exptl. and theor.
model to describe triple pairwise cross correlations incorporating cross talk
and possible changes in emission characteristics such as quenching upon binding
are outlined. The effectiveness of SW-FCCS to detect binding of three
interacting partners is exptl. verified with a std. ligand-receptor model,
biotin-streptavidin, where differently labeled biotin ligands and their binding
to a third-color labeled streptavidin are studied. The cross-correlation
amplitudes and their changes with stoichiometric binding are analyzed and the
upper limits of dissocn. consts. are detd. Performed with appropriate neg.
controls, SW-FCCS can det. interaction patterns between ligands and receptors.
Ito, S., T. Sugiyama, N. Toitani, G.
Katayama, L. Pan, N. Tamai and H. Miyasaka. (2006) Molecular translational
diffusion in solution under radiation pressure of near infrared laser light.
Proceedings of SPIE-The International Society for Optical Engineering 6326(Optical
Trapping and Optical Micromanipulation III):632605/1-632605/8.
Fluorescence
correlation spectroscopy (FCS) was applied to investigate mol. translational
diffusion in the soln. of water, ethylene glycol, and heavy water under
gradient light field of a near IR (NIR) laser beam. The diffusion times of
Rhodamine-6G in ethylene glycol and Rhodamine-123 in water became faster with
an increase in the NIR laser power owing to absorption of the NIR light by the
solvents. We also applied the radiation pressure of the NIR laser light to
cadmium telluride (CdTe) nanoparticles dispersed in heavy water, resulting in
increase in the av. no. of the CdTe particles in the confocal vol. with
increasing the NIR laser power.
Iyer, V., M. J. Rossow and M. N.
Waxham. (2006) Peak two-photon molecular brightness of fluorophores is a
robust measure of quantum efficiency and photostability. Journal of the
Optical Society of America B: Optical Physics 23(7):1420-1433.
To
date, the suitability of a fluorophore for applications involving 2-photon
absorption has generally been characterized by its 2-photon cross-section. Here
the authors consider the robustness and significance of an alternative measure
termed the mol. brightness-the fluorescence emission per mol.-which can be
obtained readily using photon-counting techniques such as fluorescence
correlation spectroscopy. The peak mol. brightness attained with increasing
excitation intensity is a reliable benchmark for various fluorescent dye solns.
This figure of merit is considered both theor. and exptl. and is related to the
2-photon quantum efficiency and the photostability properties of a dye soln.,
while it is independent of the soln.'s 2-photon cross section. This benchmark
carries considerable practical as well as scientific interest.
Jung, C., B. K. Mueller, D. C. Lamb,
F. Nolde, K. Muellen and C. Braeuchle. (2006) A New Photostable Terrylene
Diimide Dye for Applications in Single Molecule Studies and Membrane Labeling.
Journal of the American Chemical Society 128(15):5283-5291.
A
new terrylene diimide-based dye (WS-TDI) that is sol. in water has been
synthesized, and its photophys. properties are characterized. WS-TDI forms
nonfluorescing H-aggregates in water that show absorption bands being
blue-shifted with respect to those of the fluorescing monomeric form. The ratio
of monomeric WS-TDI to aggregated WS-TDI was detd. to be 1 in 14 400 from
fluorescence correlation spectroscopy (FCS) measurements, suggesting the
presence of a large amt. of sol., nonfluorescent aggregates in water. The
presence of a surfactant such as Pluronic P123 or CTAB leads to the disruption
of the aggregates due to the formation of monomers in micelles. This is
accompanied by a strong increase in fluorescence. A single mol. study of WS-TDI
in polymeric films of PVA and PMMA reveals excellent photostability with
respect to photobleaching, far above the photostability of other common
water-sol. dyes, such as oxazine-1, sulforhodamine-B, and a water-sol.
perylenediimide deriv. Furthermore, labeling of a single protein such as avidin
is demonstrated by FCS and single mol. photostability measurements. The high
tendency of WS-TDI to form nonfluorescent aggregates in water in connection
with its high affinity to lipophilic environments is used for the fluorescence
labeling of lipid membranes and membrane contg. compartments such as artificial
liposomes or endosomes in living HeLa cells. The superior fluorescence imaging
quality of WS-TDI in such applications is demonstrated in comparison to other
well-known membrane staining dyes such as Alexa647 conjugated with dextran and
FM 4-64 lipophilic styryl dye.
Jung, G. and A. Zumbusch. (2006) Improving
autofluorescent proteins: comparative studies of the effective brightness of
green fluorescent protein (GFP) mutants. Microscopy Research and Technique 69(3):175-185.
We
study the photophys. behavior of 8 mutants of Green Fluorescent Protein (GFP)
using fluorescence correlation spectroscopy (FCS) on the single mol. level and
double resonance excitation of bulk samples. Exptl. data reported here and the
previously published data on the RH/R- equil. and fluorescence quantum yields
FFl are analyzed with respect to single mol. as well as conventional
fluorescence microscopy. The fraction of GFP mols. in a dark state, [D],
reduces the effective absorption cross section under photostationary
conditions. The detn. of the excitable fraction [B] and its fluorescence
quantum yield FFl gives the effective brightness Feff. Our results show that in
its wavelength range, eGFP is, among the GFPs, the best fluorophore for most
microscopic applications. However, in the red shifted YFP-proteins, there is
still potential for improvement, since a pronounced dark state population is
detectable in all mutants investigated so far. We propose to use the mutant
T203Y/E222Q in imaging studies, whenever the expression yield is not a limiting
factor. In FCS expts., where the useful concn. range of the expressed mols. is
restricted to concns. below micromolarity, our data suggest the use of wt-GFP
or mutant T203Y, as these represent photochem. buffers. Both mutants might
surpass the limitations given by out-of-focus bleaching in live cell
microscopy.
Kahya, N. (2006) Targeting
membrane proteins to liquid-ordered phases: molecular self-organization
explored by fluorescence correlation spectroscopy. Chemistry and Physics of
Lipids 141(1-2):158-168.
A
review. The complex and dynamic architecture of biol. membranes comprises of
various heterogeneities, some of which may include lipid-based and/or
protein-based microdomains called \"rafts\". Due to interactions
among membrane components, several types of domains can form with different
characteristics and mechanisms of formation. Model membranes, such as giant
unilamellar vesicles (GUVs), provide a key system to study lipid-lipid and
lipid-protein interactions, which are potentially relevant to raft formation,
by (single-mol.) optical microscopy. Here, we review studies of combined
confocal imaging and fluorescence correlation spectroscopy (FCS) on lipid
dynamics and organization in domains assembled in GUVs, prepd. from various
lipid mixts., which are relevant to the problem of raft formation. Finally, we
summarize the results on lipid-protein interactions, which govern the targeting
of several putative raft- and non-raft-assocd. membrane proteins to
domain-exhibiting GUVs.
Kahya, N. and P. Schwille. (2006) Fluorescence
correlation studies of lipid domains in model membranes (Review). Molecular
Membrane Biology 23(1):29-39.
A
review. Advances in optical microscopy techniques and single-mol. detection
have paved the way to exploring new approaches for investigating membrane
dynamics and organization, thereby revealing details on the processing of
signals, complex assocn./dissocn., chem. reactions and transport at and around
the membrane. These events rely on a tight regulation of lipid-protein and
protein-protein interactions in space and time. Fluorescence Correlation
Spectroscopy (FCS) provides exquisite sensitivity in measuring local concns.,
assocn./dissocn. consts., chem. rate consts. and, in general, in probing the
chem. environment of the species of interest and its interactions with
potential partners. Here, we review some applications of FCS to lipid and
protein organization in biomimetic membranes with lateral heterogeneities,
which share some physico-chem. properties with cellular rafts. What we learn
from investigations of lipid-lipid and lipid-protein interactions in simple
model membranes can be regarded as an essential basic lecture for studies in
more complex cellular membranes.
Kahya, N. and P. Schwille. (2006) How
Phospholipid-Cholesterol Interactions Modulate Lipid Lateral Diffusion, as
Revealed by Fluorescence Correlation Spectroscopy. Journal of Fluorescence 16(5):671-678.
Cholesterol
is a key player in regulating physico-chem. properties of cellular membranes
and, thereby, ensuring cell viability. In particular, lipid-cholesterol
interactions may provide important information on the spatio-temporal organization
of membrane components. Here, the authors apply confocal imaging and
Fluorescence Correlation Spectroscopy (FCS) to Giant Unilamellar Vesicles
(GUVs) composed of binary mixts. of lipids and cholesterol. The effect of
cholesterol on lipid dynamics and mol. packing order of unsatd., monounsatd.,
fully satd. (with both low and high phase transition temps., Tm)
glycero-phospholipids and sphingomyelin was investigated. The authors show
that, for unsatd. glycerophospholipids, the decrease of the lipid diffusion
coeff. as a result of the interaction with cholesterol does not depend on the
fatty acid chain length. However, the values of the diffusion coeff. change as
a function of chain length. The monounsatd. phospholipid
palmitoyl-oleoyl-phosphatidylcholine (POPC) exhibits a dynamic behavior very
similar to the unsatd. dioleoyl-phosphatidylcholine (DOPC). By contrast, for
satd. (low Tm) glycero-phospholipids, cholesterol causes a decrease of lipid
mobility in a chain length-dependent manner. FCS can be employed as a valuable
tool to study lipid-sterol interactions and their effect on lipid dynamics,
mol. packing and degree of conformational order.
Kang, K., A. Wilk, J. Buitenhuis, A.
Patkowski and J. K. G. Dhont. (2006) Diffusion of spheres in isotropic and
nematic suspensions of rods. Journal of Chemical Physics 124(4):044907/1-044907/17.
Diffusion
of a small tracer sphere (apoferritin) in isotropic and nematic networks [of fd
virus] is discussed. For a tracer sphere that is smaller than the mesh size of
the network, screened hydrodynamic interactions between the sphere and the
network det. its diffusion coeff. A theory is developed for such interactions
as well as their relation to the long-time self-diffusion coeff. Fluorescence
correlation spectroscopy measurements on mixts. of apoferritin and fd virus are
presented. The long-time self-diffusion coeff. of apoferritin is measured as a
function of the fd-virus concn., both in the isotropic and nematic state, in
directions parallel and perpendicular to the nematic director. The hydrodynamic
screening length of the fd-virus network as a function of fd concn. is obtained
by combining these exptl. data with the theory. Surprisingly, the screening
length increases with increasing concn. in nematic networks. This is due to the
increase in the degree of alignment, which apparently leads to a strong
increase of the screening length. Hydrodynamic screening is thus strongly
diminished by alignment. A self-consistent calcn. of the screening length does
not work at higher concns., probably due to the strong variation of the typical
incident flow fields over the contour of a rod.
Kannan, B., J. Y. Har, P. Liu, I.
Maruyama, J. L. Ding and T. Wohland. (2006) Electron Multiplying
Charge-Coupled Device Camera Based Fluorescence Correlation Spectroscopy.
Analytical Chemistry 78(10):3444-3451.
A
fluorescence correlation spectroscopy (FCS) setup is built with an electron
multiplying charge-coupled device camera. Although the instrument has a limited
time resoln. of 4 ms, compared to 0.1-0.2 ms for common instruments using
avalanche photodiodes, it allows multiplexing of FCS measurements, has a
software-adjustable pinhole after data collection, performs flow speed as well
as flow direction measurements in microchannels and could be used to do
spectral FCS. Measurements are performed on fluorescent dyes and polystyrene
beads in high-viscosity media and on epidermal growth factor receptors in
Chinese hamster ovary cells. Using real measurements on single spots,
multiplexing of focal spots and detection elements are simulated and the
results are discussed.
Kawai-Noma, S., S. Ayano, C.-G.
Pack, M. Kinjo, M. Yoshida, K. Yasuda and H. Taguchi. (2006) Dynamics of
yeast prion aggregates in single living cells. Genes to Cells 11(9):1085-1096.
Prions
are propagating proteins that are ordered protein aggregates, in which the
phenotypic trait is retained in the altered protein conformers. To understand
the dynamics of the prion aggregates in living cells, we directly monitored the
fate of the aggregates using an on-chip single-cell cultivation system as well
as fluorescence correlation spectroscopy (FCS). Single-cell imaging revealed
that the visible foci of yeast prion Sup35 fused with GFP are dispersed
throughout the cytoplasm during cell growth, but retain the prion phenotype.
FCS showed that [PSI+] cells, irresp. of the presence of foci, contain diffuse
oligomers, which are transmitted to their daughter cells. Single-cell
observations of the oligomer-based transmission provide a link between previous
in vivo and in vitro analyses of the prion and shed light on the relationship
between the protein conformation and the phenotype.
Kim, J., S. Doose, H. Neuweiler and
M. Sauer. (2006) The initial step of DNA hairpin folding: a kinetic analysis
using fluorescence correlation spectroscopy. Nucleic Acids Research 34(9):2516-2527.
Conformational
fluctuations of single-stranded DNA (ssDNA) oligonucleotides were studied in
aq. soln. by monitoring contact-induced fluorescence quenching of the oxazine
fluorophore MR121 by intrinsic guanosine residues (dG). We applied fluorescence
correlation spectroscopy as well as steady-state and time-resolved fluorescence
spectroscopy to analyze kinetics of DNA hairpin folding. We first characterized
the reporter system by investigating bimol. quenching interactions between
MR121 and guanosine monophosphate in aq. soln. estg. rate consts., efficiency
and stability for formation of quenched complexes. We then studied the kinetics
of complex formation between MR121 and dG residues site-specifically
incorporated in DNA hairpins. To uncover the initial steps of DNA hairpin
folding we investigated complex formation in ssDNA carrying one or two
complementary base pairs (dC-dG pairs) that could hybridize to form a short
stem. Our data show that incorporation of a single dC-dG pair leads to
non-exponential decays for opening and closing kinetics and reduces rate
consts. by one to two orders of magnitude. We found pos. activation enthalpies
independent of the no. of dC-dG pairs. These results imply that the rate
limiting step of DNA hairpin folding is not detd. by loop dynamics, or by
mismatches in the stem, but rather by interactions between stem and loop
nucleotides.
Kitamura, A., H. Kubota, C.-G. Pack,
G. Matsumoto, S. Hirayama, Y. Takahashi, H. Kimura, M. Kinjo, R. I. Morimoto
and K. Nagata. (2006) Cytosolic chaperonin prevents polyglutamine toxicity
with altering the aggregation state. Nature Cell Biology 8(10):1163-1169.
Polyglutamine
(polyQ)-expansion proteins cause neurodegenerative disorders including
Huntington's disease, Kennedy's disease and various ataxias. The cytotoxicity
of these proteins is assocd. with the formation of aggregates or other
conformationally toxic species. Here, we show that the cytosolic chaperonin CCT
(also known as TRiC) can alter the course of aggregation and cytotoxicity of
huntingtin (Htt)-polyQ proteins in mammalian cells. Disruption of the CCT
complex by RNAi-mediated knockdown enhanced Htt-polyQ aggregate formation and
cellular toxicity. Anal. of the aggregation states of the Htt-polyQ proteins by
fluorescence correlation spectroscopy revealed that CCT depletion results in
the appearance of sol. Htt-polyQ aggregates. Similarly, overexpression of all
eight subunits of CCT suppressed Htt aggregation and neuronal cell death. These
results indicate that CCT has an essential role in protecting against the
cytotoxicity of polyQ proteins by affecting the course of aggregation.
Kosturko, L. D., M. J. Maggipinto,
G. Korza, J. W. Lee, J. H. Carson and E. Barbarese. (2006) Heterogeneous
nuclear ribonucleoprotein (hnRNP) E1 binds to hnRNP A2 and inhibits translation
of A2 response element mRNAs. Molecular Biology of the Cell 17(8):3521-3533.
Heterogeneous
nuclear ribonucleoprotein (hnRNP) A2 is a trans-acting RNA-binding protein that
mediates trafficking of RNAs contg. the cis-acting A2 response element (A2RE).
Previous work has shown that A2RE RNAs are transported to myelin in
oligodendrocytes and to dendrites in neurons. HnRNP E1 is an RNA-binding
protein that regulates translation of specific mRNAs. Here, we show by yeast
two-hybrid anal., in vivo and in vitro coimmunopptn., in vitro crosslinking,
and fluorescence correlation spectroscopy that hnRNP E1 binds to hnRNP A2 and
is recruited to A2RE RNA in an hnRNP A2-dependent manner. HnRNP E1 is
colocalized with hnRNP A2 and A2RE mRNA in granules in dendrites of
oligodendrocytes. Overexpression of hnRNP E1 or microinjection of exogenous
hnRNP E1 in neural cells inhibits translation of A2RE mRNA, but not of non-A2RE
RNA. Excess hnRNP E1 added to an in vitro translation system reduces
translation efficiency of A2RE mRNA, but not of nonA2RE RNA, in an hnRNP
A2-dependent manner. These results are consistent with a model where hnRNP E1
recruited to A2RE RNA granules by binding to hnRNP A2 inhibits translation of
A2RE RNA during granule transport.
Kral, T., M. Langner and M. Hof.
(2006) DNA-Spermine and DNA-Lipid Aggregate Formation Visualized by
Fluorescence Correlation Spectroscopy. Chemotherapy (Basel, Switzerland) 52(4):196-199.
Background:
Fluorescence correlation spectroscopy (FCS) can be used for the detn. of
diffusion coeffs. of single mols. Since diffusion coeffs. are correlated with
size and shape of the labeled species, FCS provides information on
conformational changes in plasmids aggregates. Methods: A 10-kbp plasmid
stained with PicoGreen was condensed by spermine or liposomes formulated from
cationic lipid and egg phosphatidylcholine. Results: The diffusion coeff. of
DNA increases from 1.0*10-12 m2/s to 3.2*10-12 m2/s by the addn. of spermine,
whereas the addn. of cationic liposomes leads to complexes characterized by
diffusion coeffs. with values ranging from 1.7 to 1.9*10-12 m2/s. Conclusions:
FCS expts. allow detg. the diffusion coeffs. of DNA-contg. aggregates which
provide information regarding the topol. and homogeneity of the aggregate.
Kyoung, M. and E. D. Sheets. (2006) Manipulating
and probing the spatio-temporal dynamics of nanoparticles near surfaces.
Proceedings of SPIE-The International Society for Optical Engineering 6326(Optical
Trapping and Optical Micromanipulation III):63262L/1-63262L/8.
In
this report, we combine total internal reflection-fluorescence correlation
spectroscopy (TIR-FCS) with a single optical trap to simultaneously manipulate
and measure the dynamics of individual mols. near the substrate-soln.
interface. As a proof of principle, polystyrene particles (84 nm in diam.) are
used as a model system to test our approach in studying their diffusion
properties near surfaces, which are treated with polyethylene glycol 8000,
bovine serum albumin or sodium hydroxide. The evanescent field of 543 nm
excitation propagates .apprx.100 nm into the soln., and the fluorescence
detection is spatially confined by a 25 or 50 mm pinhole that is parfocal with
the specimen plane. The optical trap is generated using a cw Ti:sapphire laser
at 780 nm. Our results indicate that the particles' diffusion is influenced by
surface interactions, which might have further implications on biomembrane
studies. Furthermore, the obsd. translational diffusion of individual particles
can be manipulated using an optical trap. By combining the single mol.
sensitivity of TIR-FCS with a noninvasive manipulation method, such as optical
trapping, we will be able to probe mol. dynamics in biomimetic systems and
living cells.
Laguecir, A., S. Ulrich, J. Labille,
N. Fatin-Rouge, S. Stoll and J. Buffle. (2006) Size and pH effect on electrical
and conformational behavior of poly(acrylic acid): Simulation and experiment.
European Polymer Journal 42(5):1135-1144.
Monte
Carlo simulations, exptl. titrns. and fluorescence correlation spectroscopy
expts. were used to investigate the conformational and elec. properties of
polyacrylic acids (PAA). On the one hand, titrn. curves were calcd. to get an
insight into the role of pH on the degree of ionization and conformation of PAA
chains. On the other hand, exptl. potentiometric titrns. of PAA were also
achieved for different PAA mol. wts. and compared to the calcd. titrn. curves
obtained by Monte Carlo coarse grained simulations. It was found that for a
large range at intermediate PAA ionizations, a good correlation is obtained
between exptl. and simulations data thanks to the prominence of electrostatic
interactions in this domain. The effect of ionic concn. and PAA mol. wt. on the
titrn. curves was also investigated. In order to get a better understanding of
PAA conformational behavior, we also investigated PAA diffusion properties in
aq. solns. as a function of pH and ionic strength by fluorescence correlation
spectroscopy (FCS), thanks to its high sensitivity to measure diffusion coeffs.
of tracer solutes. Good qual. agreements were obsd. between exptl.
diffusivities and polymer properties calcd. from MC simulations. It was shown
that the high mol. wt. PAA chains display more significant changes in
diffusivity in agreement with the ionization degrees and conformational changes
obsd. in the simulations.
Le, T. T., T. Emonet, S. Harlepp, C.
C. Guet and P. Cluzel. (2006) Dynamical determinants of drug-inducible gene
expression in a single bacterium. Biophysical Journal 90(9):3315-3321.
A
primitive example of adaptation in gene expression is the balance between the
rate of synthesis and degrdn. of cellular RNA, which allows rapid responses to
environmental signals. Here, we investigate how multidrug efflux pump systems
mediate the dynamics of a simple drug-inducible system in response to a steady
level of inducer. Using fluorescence correlation spectroscopy, we measured in
real time within a single bacterium the transcription activity at the RNA level
of the acrAB-TolC multidrug efflux pump system. When cells are exposed to
const. level of anhydrotetracycline inducer and are adsorbed onto a
poly-L-lysine-coated surface, we found that the acrAB-TolC promoter is steadily
active. We also monitored the activity of the tet promoter to characterize the
effect of this efflux system on the dynamics of drug-inducible transcription.
We found that the transcriptional response of the tet promoter to a steady
level of aTc rises and then falls back to its preinduction level. The rate of
RNA degrdn. was const. throughout the transcriptional pulse, indicating that
the modulation of intracellular inducer concn. alone can produce this pulsating
response. Single-cell expts. together with numerical simulations suggest that
such pulsating response in drug-inducible genetic systems is a property
emerging from the dependence of drug-inducible transcription on multidrug
efflux systems.
Lenne, P.-F., L. Wawrezinieck, F.
Conchonaud, O. Wurtz, A. Boned, X.-J. Guo, H. Rigneault, H.-T. He and D.
Marguet. (2006) Dynamic molecular confinement in the plasma membrane by
microdomains and the cytoskeleton meshwork. EMBO Journal 25(14):3245-3256.
It
is by now widely recognized that cell membranes show complex patterns of
lateral organization. Two mechanisms involving either a lipid-dependent
(microdomain model) or cytoskeleton-based (meshwork model) process are thought
to be responsible for these plasma membrane organizations. In the present
study, fluorescence correlation spectroscopy measurements on various spatial
scales were performed in order to directly identify and characterize these two
processes in live cells with a high temporal resoln., without any loss of
spatial information. Putative raft markers were found to be dynamically
compartmented within tens of milliseconds into small microdomains ([Character
Omitted]<120 nm) that are sensitive to the cholesterol and sphingomyelin
levels, whereas actin-based cytoskeleton barriers are responsible for the
confinement of the transferrin receptor protein. A free-like diffusion was
obsd. when both the lipid-dependent and cytoskeleton-based organizations were
disrupted, which suggests that these are two main compartmentalizing forces at
work in the plasma membrane.
Lessard, G. A., P. M. Goodwin and J.
H. Werner. (2006) Three-dimensional tracking of fluorescent particles.
Proceedings of SPIE-The International Society for Optical Engineering 6092(Ultrasensitive
and Single-Molecule Detection Technologies):609205/1-609205/8.
Single
mol. measurements are generally made in conditions that depart from physiol.
conditions, such as with mols. excised from cells or even immobilized on
surfaces. Such departures can easily cause measurements on biomols. to be
inexact. A tracking instrument to follow a single mol.'s path in three
dimensions inside a living cell would be a major step towards enabling
single-mol. observations in physiol. conditions. We describe an instrument that
will extend the state of the art in single-mol. tracking technol., allowing
extended observations of single particles as they diffuse and are transported.
Computations show that our approach should be capable of tracking a
protein-sized object diffusing at intracellular speeds for av. times of over
two seconds - long enough to track a typical fluorescent mol. from capture to
photobleaching.
Leutenegger, M., M. Gosch, A. Perentes,
P. Hoffmann, O. J. F. Martin and T. Lasser. (2006) Confining the sampling
volume for fluorescence correlation spectroscopy using a sub-wavelength sized
aperture. Optics Express 14(2):956-969.
For
the observation of single mol. dynamics with fluorescence fluctuation
spectroscopy (FFS) very low fluorophore concns. are necessary. For in vitro
measurements, this requirement is easy to fulfill. In biol. however, micromolar
concns. are often encountered and may pose a real challenge to conventional FFS
methods based on confocal instrumentation. We show a higher confinement of the
sampling vol. in the near-field of sub-wavelength sized apertures in a thin
gold film. The gold apertures have been measured and characterized with
fluorescence correlation spectroscopy (FCS), indicating light confinement
beyond the far-field diffraction limit. We measured a redn. of the effective
sampling vol. by an order of magnitude compared to confocal instrumentation.
Li, Q. and S. Seeger. (2006) Label-Free
Detection of Single Protein Molecules Using Deep UV Fluorescence Lifetime
Microscopy. Analytical Chemistry 78(8):2732-2737.
The
authors present the detection of single b-galactosidase mols. from Escherichia
coli (Ecb Gal) using deep UV laser-based fluorescence lifetime microscopy. The
native fluorescence from intrinsic tryptophan emission has been obsd. after
one-photon excitation at 266 nm. Applying the time-resolved single-photon
counting method, the authors investigated the fluorescence lifetime
distribution and the bursts of autofluorescence photons from tryptophan
residues in Ecb Gal protein as well as fluorescence correlation spectroscopy of
Ecb Gal. The results demonstrate that deep UV laser-based fluorescence lifetime
microscopy is useful for identification of biol. macromols. at the single-mol.
level using intrinsic fluorescence.
Lill, Y., M. A. Lill, B. Fahrenkrog,
K. Schwarz-Herion, S. Paulillo, U. Aebi and B. Hecht. (2006) Single
hepatitis-B virus core capsid binding to individual nuclear pore complexes in
HeLa cells. Biophysical Journal 91(8):3123-3130.
We
investigate the interaction of hepatitis B virus capsids lacking a nuclear
localization signal with nuclear pore complexes (NPCs) in permeabilized HeLa
cells. Confocal and wide-field optical images of the nuclear envelope show
well-spaced individual NPCs. Specific interactions of capsids with single NPCs
are characterized by extended residence times of capsids in the focal vol.
which are characterized by fluorescence correlation spectroscopy. In addn., single-capsid-tracking
expts. using fast wide-field fluorescence microscopy at 50 frames/s allow us to
directly observe specific binding via a dual-color colocalization of capsids
and NPCs. We find that binding occurs with high probability on the nuclear-pore
ring moiety, at 44 +- 9 nm radial distance from the central axis.
Liu, Y., H.-R. Kim and A. A. Heikal.
(2006) Structural Basis of Fluorescence Fluctuation Dynamics of Green
Fluorescent Proteins in Acidic Environments. Journal of Physical Chemistry
B 110(47):24138-24146.
Green
fluorescent proteins (GFPs) have become powerful markers for numerous biol.
studies due to their robust fluorescence properties, site-specific labeling, pH
sensitivity, and mutations for multiple-site labeling. Fluorescence correlation
spectroscopy (FCS) studies have indicated that fluorescence blinking of anionic
GFP mutants takes place on a time scale of 45-300 ms, depending on pH, and have
been attributed to external proton transfer. Here we present exptl. evidence
indicating that conformational change in the protein b-barrel is a detg. step
for the external protonation of GFP-S65T (at low pH) using time-resolved
fluorescence and polarization anisotropy measurements. While the av. anionic
fluorescence lifetime of GFP-S65T is reduced by .apprx.18% over a pH range of
3.6-10.0, the fluorescence polarization anisotropy decays mostly as a single
exponential with a rotational time of j = 17+-1 ns, which indicates an intact
b-barrel with a hydrodynamic vol. of 78+-5 nm3. In contrast, the total
fluorescence (525+-50 nm) of the excited neutral state of S65T reveals a strong
correlation between the fluorescence lifetime, structural conformation, and pH.
The av. fluorescence lifetime of the excited neutral state of S65T as a
function of pH yields pKa ~ 5.9 in agreement with literature values using
steady-state techniques. In contrast to the intact b-barrel at high pH, the
anisotropy of neutral S65T (at pH ? pKa) decays as a biexponential (e.g., at pH
5.8, j1 = 1.86 ns, b1 = 0.03, j2 = 17.5 ns, and b2 = 0.25), which suggests a
segmental mobility of the chromophore assocd. with conformational changes of
the protein. The segmental motion of the S65T chromophore becomes faster with
an enhanced amplitude ratio as pH is reduced. For comparative purposes, we also
provide complementary FCS results on fluorescence blinking of the excited
neutral state of an EGFP mutant (F64L/S65T) on a much slower time scale. Our
results indicate that conformational rearrangement of the b-barrel and the
amino acids surrounding the embedded chromophore is a rate-detg. step for
external proton transfer and possibly cis/trans isomerization as nonradiative
pathways that underlie fluorescence blinking of GFP mutants in an acidic
environment. In addn., the neutral-state transition is likely to be involved in
the blinking process previously obsd. for the anionic-state transition in
several GFP mutants.
Magzoub, M., P. Padmawar, J. A. Dix
and A. S. Verkman. (2006) Millisecond Association Kinetics of K+ with
Triazacryptand-Based K+ Indicators Measured by Fluorescence Correlation
Spectroscopy. Journal of Physical Chemistry B 110(42):21216-21221.
The
authors recently introduced a water-sol., long-wavelength K+-sensing indicator,
TAC-Red, consisting of a triazacryptand K+-selective ionophore coupled to a
xanthylium chromophore (Nat. Methods 2005, 2, 825-827). Stopped-flow kinetic
anal. indicated that in response to changes in K+ concn. TAC-Red fluorescence
enhancement occurs in milliseconds or less. Here, the authors use fluorescence
correlation spectroscopy to quantify the binding kinetics of K+ with TAC-Red
and a new, longer-wavelength sensor, TAC-Crimson. Autocorrelation functions,
G(t), were similar at 0 and high (150 mM) K+ concns., with the appearance of a
prominent kinetic process with a correlation time in the millisecond range for
K+ concns. between .apprx.20 and 60 mM. Control expts. with increased
illumination vol. and soln. viscosity indicated that the millisecond component
represented K+/TAC-Red assocn. K+-dependent G(t) data, modeled using a global
regression to a binding/diffusion model, gave assocn. and dissocn. rate consts.
of 0.0020+-0.0003 mM-1 ms-1 and 0.12+-0.02 ms-1, resp., for TAC-Red. Similar
results were obtained for TAC-Crimson. The rapid K+ binding kinetics with
triazacryptand-based sensors support their utility for measuring changes in K+
concns. during rapid neural signaling and ion channel gating.
Masuda, A., K. Ushida and T.
Okamoto. (2006) New fluorescence correlation spectroscopy (FCS) suitable for
the observation of anomalous diffusion in polymer solution: Time and space
dependences of diffusion coefficients. Journal of Photochemistry and
Photobiology, A: Chemistry 183(3):304-308.
The
authors propose a new method of fluorescence correlation spectroscopy (FCS)
enabling direct detection of anomalous diffusion as the distance dependence of
diffusion coeffs. (DDDC), namely, sampling-vol.-controlled (SVC)-FCS. In
discussing the results of SVC-FCS measurement of mol. diffusion in aq.
hyaluronan (HA) solns. in this paper, the authors we suggest the use of
\"local anomalous diffusion\" based on the differential of the
mean-square displacement (MSD) which is convenient for understanding the total
lineshapes of the obsd. diffusion coeff. as a function of the diffusion
distance or the diffusion time.
Matsunaga, S., N. Ohmido and K.
Fukui. (2006) Chromosome dynamics in tobacco BY-2 cultured cells.
Biotechnology in Agriculture and Forestry 58(Tobacco BY-2 Cells):51-63.
A
review. Bright Yellow-2 (BY-2) cultured cells are suitable for analyses of
chromosome dynamics during mitosis because of their chromosome size and short
cell cycle. Chromosome dynamic analyses using tobacco BY-2 cells is
particularly well suited to applications of advanced imaging analyses including
fluorescence recovery after photobleaching, fluorescence correlation
spectroscopy, and fluorescence resonance energy transfer.
Meacci, G., J. Ries, E.
Fischer-Friedrich, N. Kahya, P. Schwille and K. Kruse. (2006) Mobility of
Min-proteins in Escherichia coli measured by fluorescence correlation
spectroscopy. Physical Biology 3(4):255-263.
In
the bacterium Escherichia coli, selection of the division site involves
pole-to-pole oscillations of the proteins MinD and MinE. Different oscillation
mechanisms based on cooperative effects between Min-proteins and on the
exchange of Min-proteins between the cytoplasm and the cytoplasmic membrane
have been proposed. The parameters characterizing the dynamics of the Min-proteins
in vivo are not known. It has therefore been difficult to compare the models
quant. with expts. Here, the authors present in vivo measurements of the
mobility of MinD and MinE using fluorescence correlation spectroscopy. Two
distinct timescales are clearly visible in the correlation curves. While the
faster timescale can be attributed to cytoplasmic diffusion, the slower
timescale could result from diffusion of membrane-bound proteins or from
protein exchange between the cytoplasm and the membrane. The authors det. the
diffusion const. of cytoplasmic MinD to be approx. 16 mm2 s-1, while for MinE
the authors find about 10 mm2 s-1, independently of the processes responsible
for the slower time-scale. The implications of the measured values for the oscillation
mechanism are discussed.
Meissner, O. and H. Haeberlein.
(2006) Influence of xanthohumol on the binding behavior of GABAA receptors
and their lateral mobility at hippocampal neurons. Planta Medica 72(7):656-658.
The
influence of the xanthohumol from Humulus lupulus L. on the binding of
muscimol-Alexa Fluor 532 (Mu-Alexa), a fluorescently labeled GABAA receptor
agonist, was studied by fluorescence correlation spectroscopy. An incubation of
hippocampal neurons with 75 nM of xanthohumol increased the specific Mu-Alexa
binding by approx. 17%, which was selectively found in GABAA receptor Mu-Alexa
complexes with hindered lateral mobility [Dbound2 = (0.11+-0.03) mm2/s] as
described with midazolam.
Miller, A. E., A. J. Fischer, T.
Laurence, C. W. Hollars, R. J. Saykally, J. C. Lagarias and T. Huser. (2006) Single-molecule
dynamics of phytochrome-bound fluorophores probed by fluorescence correlation
spectroscopy. Proceedings of the National Academy of Sciences of the United
States of America 103(30):11136-11141.
Fluorescence
correlation spectroscopy (FCS) was used to investigate the hydrodynamic and
photophys. properties of PR1 (phytofluor red 1), an intensely red fluorescent
biliprotein variant of the truncated cyanobacterial phytochrome 1 (Cph1D, which
consists of the N-terminal 514 amino acids). Single-mol. diffusion measurements
showed that PR1 has excellent fluorescence properties at the single-mol. level,
making it an interesting candidate for red fluorescent protein fusions. FCS
measurements for probing dimer formation in soln. over a range of protein
concns. were enabled by addn. of Cph1D apoprotein (apoCph1D) to nanomolar
solns. of PR1. FCS brightness anal. showed that heterodimerization of PR1 with
apoCph1D altered the chem. environment of the PR1 chromophore to further
enhance its fluorescence emission. Fluorescence correlation measurements also
revealed interactions between apoCph1D and the red fluorescent dyes Cy5.18 and
Atto 655 but not Alexa Fluor 660. The concn. dependence of protein:dye complex
formation indicated that Atto 655 interacted with, or influenced the formation
of, the apoCph1 dimer. These studies presage the utility of phytofluor tags for
probing single-mol. dynamics in living cells in which the fluorescence signal
can be controlled by the addn. of various chromophores that have different
structures and photophys. properties, thereby imparting different types of
information, such as dimer formation or the presence of open binding faces on a
protein.
Noel, S., J. Buffle, N. Fatin-Rouge
and J. Labille. (2006) Factors affecting the flux of macromolecular, labile,
metal complexes at consuming interfaces, in water and inside agarose gel: SSCP
study and environmental implications'. Journal of Electroanalytical
Chemistry 595(2):125-135.
In
environmental processes, the contribution of macromol. or colloidal metal
complexes to the overall diffusion flux of metal may be of major importance.
For labile complexes, the computation of such flux is based on the concept of
av. diffusion coeff., which has only been checked systematically with small
size ligands and in aq. soln. This concept is checked here, both in aq. soln.
and in agarose hydrogel, with large size macromols.: dextrans of molar masses
10,000, 40,000 and 464,000, modified to introduce fluorescent or complexing
(aspartate) moieties in their structure were used. Their diffusion properties
in H2O and agarose gel are detd. by fluorescence correlation spectroscopy
(FCS). The complexing and diffusive properties of their Pb(II) complexes in H2O
and agarose hydrogel are also detd. by scanning stripping chronopotentiometry
(SSCP). Both in H2O and gel, the concept of av. diffusion coeff. is applicable
to compute the flux of these labile complexes at consuming interfaces. Also the
3 ligands and their Pb-complexes diffuse as spherical coils, in H2O and in the
gel, in accordance with other recent results. The implications of these
results, in environmental applications, in particular in biouptake, and for the
development of in situ sensors for environmental monitoring, are discussed, as
well as the optimum conditions of voltammetric measurements with gel integrated
microelectrode (GIME) in natural media contg. colloidal complexes. In
particular provided the gel is correctly preequilibrated with the test soln.,
and its thickness is larger than 150 mm, diffusion/reaction in the test soln.
do not influence the voltammetric flux at microelectrodes in the gel. The same
conclusion is applicable to microorganisms inside biofilms.
Noguchi, E., Y. Ohtsuki, K.
Tokunaga, M. Yamaoka-Sageshima, K. Ichikawa, T. Aoki, M. Shibasaki and T.
Arinami. (2006) ADAM33 polymorphisms are associated with asthma
susceptibility in a Japanese population. Clinical and Experimental Allergy 36(5):602-608.
Background:
Asthma is the most common chronic disorder in childhood, and asthma
exacerbation is an important cause of childhood morbidity and hospitalization.
Asthma is believed to be a complex disorder involving genetic and environmental
factors, and several asthma susceptibility loci have been identified through
genome-wide screening. A disintegrin and metalloprotease 33 (ADAM33) was the
first asthma susceptibility gene to be discovered by positional cloning in
2002. Objective: The aim of the present study was to investigate whether
single-nucleotide polymorphisms (SNPs) in ADAM33 are assocd. with childhood
asthma in the Japanese population. Methods: Twenty-three ADAM33 SNPs were
genotyped by fluorescence correlation spectroscopy with the use of DNA from 155
families (538 members) identified through children with atopic asthma. The
transmission disequil. test (TDT) was performed for family-based assocn. study.
Results TDT revealed that minor alleles of S + 1, ST + 4, and T2 SNPs were
over-transmitted to asthma-affected offspring (P < 0.05). According to the
haplotype TDT, no haplotype of ADAM33 was transmitted preferentially to
asthmatic offspring. Conclusion: Our results confirm the involvement of ADAM33
in the development of childhood asthma among the Japanese.
Nomura, Y., H. Fuchigami, H. Kii, Z.
Feng, T. Nakamura and M. Kinjo. (2006) Detection of oxidative stress-induced
mitochondrial DNA damage using fluorescence correlation spectroscopy.
Analytical Biochemistry 350(2):196-201.
Using
fluorescence correlation spectroscopy (FCS), the authors tested the feasibility
of rapid detection of oxidative damage of mitochondrial DNA (mtDNA) in a small
vol. The complete mtDNA genome was amplified by long polymerase chain reaction
(LPCR), and the product was fluorescently labeled with an intercalating dye,
YOYO-1. The fluorescence autocorrelation function was analyzed using a simple
two-component model with the diffusion time of 0.21 ms for the LPCR primer and
18 ms for the mtDNA LPCR product. When human embryonic kidney 293 (HEK-293) cells
were exposed to 0.4 mM H2O2, the fraction of the mtDNA LPCR product decreased
significantly. In contrast, the fraction of the nuclear-encoded b-globin LPCR
product remained unchanged. The anal. time of FCS measurement was very short (5
min) compared with that of gel electrophoresis (3 h). Thus, FCS allowed the
rapid detection of the vulnerability of mtDNA to oxidative stress within a
small vol. element at the subfemtoliter level in soln. These results suggest
that the LPCR-FCS method can be used for epidemiol. studies of diseases caused
by mtDNA damage.
Nomura, Y., H. Fuchigami, H. Kii, Z.
Feng, T. Nakamura and M. Kinjo. (2006) Quantification of size distribution
of restriction fragments in mitochondrial genome using fluorescence correlation
spectroscopy. Experimental and Molecular Pathology 80(3):275-278.
A
crucial investigation is to quantify restriction fragment length polymorphisms
without gel electrophoresis, as the distribution of fragment size is mainly
evaluated on the gel, which cannot be easily quantified. We developed a method
to det. the fragmentation of the mitochondrial genome caused by restriction
enzymes using fluorescence correlation spectroscopy (FCS). Distribution of
fragment size was evaluated by the decrease in amplitude of the fluorescence
correlation function while the mitochondrial genome PCR product was digested
with Hga I or Hae III. Using a multicomponent model, which was considered as a
fragment length-weighted correlation function, we calcd. the correlation
amplitude theor. expected and compared it to that measured by FCS. These
amplitudes for Hga I were coincident, whereas the measured amplitude for Hae
III was more than the theor. one. Because of tetra-nucleotide recognition by
Hae III, there were many more fragments than with Hga I. Therefore, the
amplitude measured by FCS would be a very useful index for primary screening
for alterations in the entire mitochondrial genome with restriction enzymes
that have several polymorphic restriction sites in the genome.
Ohsugi, Y. and M. Kinjo. (2006) Analysis
of membrane-binding protein mobility in living cells using total internal
reflection fluorescence correlation spectroscopy. Biophysical Reviews and
Letters 1(3):293-299.
Total
internal reflection fluorescence correlation spectroscopy (TIR-FCS) is an
appropriate method for measuring diffusion consts. and the no. of fluorescent
mols. very close to the coverglass surface. Recently, we have reported the
application of TIR-FCS to cell biol., measuring membrane-binding farnesylated
green fluorescent proteins (EGFP-F) in living cells. In this research, we
measured the signal transduction mol., protein kinase C (PKC), fused with EGFP
in living HeLa cells by using TIR-FCS. We obsd. two different diffusional
mobilities of PKCbII-EGFP, three-dimensional faster diffusion near the plasma
membrane and slower lateral diffusion on the plasma membrane after adenosine
tri phosphate (ATP) activation. These results indicate that it is possible to
use TIR-FCS in the study of mol. dynamics and interactions of signal
transduction proteins on the plasma membrane of the living cell.
Ohsugi, Y., K. Saito, M. Tamura and
M. Kinjo. (2006) Lateral mobility of membrane-binding proteins in living
cells measured by total internal reflection fluorescence correlation
spectroscopy. Biophysical Journal 91(9):3456-3464.
Total
internal reflection fluorescence correlation spectroscopy (TIR-FCS) allows the
authors to measure diffusion consts. and the no. of fluorescent mols. in a
small area of an evanescent field generated on the objective of a microscope.
The application of TIR-FCS makes possible the characterization of reversible
assocn. and dissocn. rates between fluorescent ligands and their receptors in
supported phospholipid bilayers. Here, for the first time, the authors extend
TIR-FCS to a cellular application for measuring the lateral diffusion of a
membrane-binding fluorescent protein, farnesylated EGFP, on the plasma
membranes of cultured HeLa and COS7 cells. The authors detected two kinds of
diffusional motion - fast three-dimensional diffusion (D1) and much slower
two-dimensional diffusion (D2), simultaneously. Conventional FCS and
single-mol. tracking confirmed that D1 was free diffusion of farnesylated EGFP
close to the plasma membrane in cytosol and D2 was lateral diffusion in the
plasma membrane. These results suggest that TIR-FCS is a powerful technique to
monitor movement of membrane-localized mols. and membrane dynamics in living
cells.
Palo, K., U. Mets, V. Loorits and P.
Kask. (2006) Calculation of photon-count number distributions via master
equations. Biophysical Journal 90(6):2179-2191.
Fitting
of photon-count no. histograms is a way of anal. of fluorescence intensity
fluctuations, a successor to fluorescence correlation spectroscopy. First versions
of the theory for calcg. photon-count no. distributions have assumed const.
emission intensity by a mol. during a counting time interval. For a long time a
question has remained unanswered: to what extent is this assumption violated in
expts.. Here the authors present a theory of photon-count no. distributions
that takes account of intensity fluctuations during a counting time interval.
Theor. count-no. distributions are calcd. via a numerical soln. of Master
equations (ME), which is a set of differential equations describing diffusion,
singlet-triplet transitions, and photon emission. Detector afterpulsing and
dead-time corrections are also included. The ME-theory is tested by fitting a
series of photon-count no. histograms corresponding to different lengths of the
counting time interval. Compared to the first version of fluorescence intensity
multiple distribution anal. theory introduced in 2000, the fit quality is
significantly improved. It is discussed how a theory of photon-count no.
distributions, which assumes const. emission intensity during a counting time
interval, may also yield a good fit quality. The authors argue that the spatial
brightness distribution used in calcns. of the fit curve is not the true
spatial brightness distribution. Instead, a no. of dynamic processes, which
cause fluorescence intensity fluctuations, are indirectly taken into account
via the profile adjustment parameters.
Pan, X., C. Aw, Y. Du, H. Yu and T.
Wohland. (2006) Characterization of poly(acrylic acid) diffusion dynamics on
the grafted surface of poly(ethylene terephthalate) films by fluorescence
correlation spectroscopy. Biophysical Reviews and Letters 1(4):433-441.
Poly(acrylic
acid) (PAAc) is a commonly used polymer grafted on poly(ethylene terephthalate)
films for the immobilization of bioactive mols. that directly interact with
living cells or tissues for the maintenance of their viability and
functionality. The diffusion property of the grafted PAAc on the surface is a
crit. parameter related to the d., length of polymer chains, and ionic strength
of the soln. Fluorescence correlation spectroscopy (FCS) is able to measure the
diffusion coeff. of fluorescent particles in soln. with single mol. sensitivity
and specificity. It was used as an effective tool to detect diffusion dynamics
of Atto 565 mols., a good indicator for viscosity of PAAc, in both aq. polymer
solns. and polymer grafted film surfaces immersed in soln. In this work we det.
the polymer chain length under different polymn. conditions in soln. and deduce
the soln. viscosity by FCS measurements of Atto 565 as tracer mol. By using the
same tracer on the grafted polymer films we can infer the viscosity of these
grafted layers under a variety of conditions, including the PAAc chain length,
the UV exposure time during polymn., the ionic strength, and the pH value of
the immersed soln.
Park, H. Y., X. Qiu, E. Rhoades, J.
Korlach, L. W. Kwok, W. R. Zipfel, W. W. Webb and L. Pollack. (2006) Achieving
Uniform Mixing in a Microfluidic Device: Hydrodynamic Focusing Prior to Mixing.
Analytical Chemistry 78(13):4465-4473.
A
microfluidic mixer is described that is well-suited for kinetic studies of
macromol. conformational change under a broad range of exptl. conditions. The
mixer exploits hydrodynamic focusing to create a thin jet contg. the macromols.
of interest. Kinetic reactions are triggered by mol. diffusion into the jet
from adjacent flow layers. The ultimate time resoln. of these devices can be
restricted by premature contact between co-flowing solns. during the focusing
process. Here, the design and characterization are described of a mixer in
which hydrodynamic focusing is decoupled from the diffusion of reactants, so
that the focusing region is free from undesirable contact between the reactants.
Uniform mixing on the microsecond time scale is demonstrated using a device
fabricated by imprinting optical-grade plastic. Device characterization is
carried out using fluorescence correlation spectroscopy (FCS) and two-photon
microscopy to measure flow speeds and to quantify diffusive mixing by
monitoring the collisional fluorescence quenching, resp. Criteria for achieving
microsecond time resoln. are described and modeled.
Perestenko, P. V. and J. M. Henley.
(2006) Visualization of AMPAR trafficking and surface expression.
Dynamic Synapse: Molecular Methods in Ionotropic Receptor Biology:119-141,, 2
plates.
A
review. The cell biol. of AMPAR synthesis, transport, targeting, and surface
expression is of crucial importance for correct neuronal function. Current
techniques and the progress achieved in the visualization of AMPAR trafficking
and surface expression are discussed. Topics covered include fluorescent
protein tools, application of XFP imaging to AMPARs, and future imaging
possibilities.
Pero, J. K., E. M. Haas and N. L.
Thompson. (2006) Size Dependence of Protein Diffusion Very Close to Membrane
Surfaces: Measurement by Total Internal Reflection with Fluorescence
Correlation Spectroscopy. Journal of Physical Chemistry B 110(22):10910-10918.
The
diffusion coeffs. of nine fluorescently labeled antibodies, antibody fragments,
and antibody complexes have been measured in soln. very close to supported
planar membranes by using total internal reflection with fluorescence
correlation spectroscopy (TIR-FCS). The hydrodynamic radii (3-24 nm) of the
nine antibody types were detd. by comparing literature values with bulk
diffusion coeffs. measured by spot FCS. The diffusion coeffs. very near
membranes decreased significantly with mol. size, and the size dependence was
greater than that predicted to occur in bulk soln. The observation that
membrane surfaces slow the local diffusion coeff. of proteins in a
size-dependent manner suggests that the primary effect is hydrodynamic as
predicted for simple spheres diffusing close to planar walls. The TIR-FCS data
are consistent with predictions derived from hydrodynamic theory. This work
illustrates one factor that could contribute to previously obsd. nonideal
ligand-receptor kinetics at model and natural cell membranes.
Piening, N., P. Weber, T. Hoegen, M.
Beekes, H. Kretzschmar and A. Giese. (2006) Photo-induced crosslinking of
prion protein oligomers and prions. Amyloid 13(2):67-77.
Prion
diseases are caused by a unique type of infectious agent, which is thought to
consist of a misfolded b-sheeted form of the a-helical cellular prion protein
(PrPC). This misfolded isoform (PrPSc) tends to form insol. amyloid-like
aggregates, impeding classical structural anal. by X-ray crystallog. or NMR.
Intermol. crosslinking may provide a means of stabilizing notoriously elusive
oligomers for further anal. and may be used for analyzing aggregate
architecture by characterizing intermol. contact sites. Using a photo-induced
crosslinking method (PICUP), aggregates of recombinant PrP (rPrP) and PrPSc
were linked at interacting surfaces via amino acid side chains. The degree of
crosslinking within PrP aggregates was adjustable using varying light
intensities and could efficiently be monitored by fluorescence correlation
spectroscopy. Specific intermol. crosslinking of PrPSc mols. was achieved even
in crude brain homogenate. Functional studies showed that stabilized aggregates
of rPrP did not loose their capacity to induce further protein aggregation and
crosslinking of PrPSc did not alter significantly the level of infectivity,
indicating that photo-induced covalent linkage of PrPSc does not destruct
surfaces important for prion propagation.
Politz, J. C. R., R. A. Tuft, K. V.
Prasanth, N. Baudendistel, K. E. Fogarty, L. M. Lifshitz, J. Langowski, D. L.
Spector and T. Pederson. (2006) Rapid, diffusional shuttling of poly(A) RNA
between nuclear speckles and the nucleoplasm. Molecular Biology of the Cell
17(3):1239-1249.
Speckles
are nuclear bodies that contain pre-mRNA splicing factors and polyadenylated
RNA. Because nuclear poly(A) RNA consists of both mRNA transcripts and
nucleus-restricted RNAs, we tested whether poly(A) RNA in speckles is dynamic
or rather an immobile, perhaps structural, component. Fluorescein-labeled
oligo(dT) was introduced into HeLa cells stably expressing a red fluorescent
protein chimera of the splicing factor SC35 and allowed to hybridize.
Fluorescence correlation spectroscopy (FCS) showed that the mobility of the
tagged poly(A) RNA was virtually identical in both speckles and at random
nucleoplasmic sites. This same result was obsd. in photoactivation-tracking
studies in which caged fluorescein-labeled oligo(dT) was used as hybridization
probe, and the rate of movement away from either a speckle or nucleoplasmic
site was monitored using digital imaging microscopy after photoactivation.
Furthermore, the tagged poly(A) RNA was obsd. to rapidly distribute throughout
the entire nucleoplasm and other speckles, regardless of whether the tracking
observations were initiated in a speckle or the nucleoplasm. Finally, in both
FCS and photoactivation-tracking studies, a temp. redn. from 37 to 22 Deg had
no discernible effect on the behavior of poly(A) RNA in either speckles or the
nucleoplasm, strongly suggesting that its movement in and out of speckles does
not require metabolic energy.
Pramanik, A. (2006) Vesicle-peptide
interactions by fluorescence correlation spectroscopy. Biochemistry and
Biophysics of Lipids:153-173.
A
review. The enterior of a phospholipid vesicle is an aq. environment. Using the
extrusion technique it is possible to prep. vesicles with different
substances/dye mols. entrapped inside. Thus, the external and internal
environment of phospholipid vesicles can be manipulated and studies can be conducted
on their ability to incorporate or to release mols., and to interact with
various substances (peptides, proteins, nucleic acids....). This review will
report how fluorescence correlation spectroscopy (FCS) at single-mol.
sensitivity can be used as a powerful biophys. tool for examg.
vesicle-peptide/protein interactions with high specificity and how
rhodamine-entrapped vesicles (REV) and rhodamine-labeled phospholipid vesicles
(RLV) can be applied to demonstrate whether a peptide/protein makes channels
into vesicle membranes or breaks vesicles. In contrast to REV, RLV do not
entrap free rhodamine inside since the fluorophore rhodamine is covalently
bound to the head groups of phospholipids. Thus, if a peptide/protein makes
channels into vesicle membranes, its exposure to vesicles will cause a release
of rhodamine only from REV but not from RLV. It is obvious that rhodamine can
not be released from RLV because the inside of RLV is free of rhodamine mols.
In contrast, if a peptide/protein breaks vesicles, its addn. to vesicles will
result in rhodamine release from both REV and RLV. As the inside of RLV is free
of rhodamine, the appearance of rhodamine in soln. will confirm that these
vesicles are broken into rhodamine-labeled phospholipid fragments. As an
example, results on mol. interactions of the peptides melittin and magainin
with REV and RLV performed by FCS will be highlighted. This study will provide
a general idea of how FCS could be used to study mol. interactions of
peptides/proteins with vesicles with special ref. to whether they form
pores/channels into vesicle membranes i.e. they are CPP (cell penetrating
peptides/proteins) or they break vesicles.
Przybylo, M., J. Sykora, J.
Humpolickova, A. Benda, A. Zan and M. Hof. (2006) Lipid Diffusion in Giant
Unilamellar Vesicles Is More than 2 Times Faster than in Supported Phospholipid
Bilayers under Identical Conditions. Langmuir 22(22):9096-9099.
The
lateral diffusion coeffs. of a BODIPY tail-labeled lipid in two model systems,
namely, free-standing giant unilamellar vesicles (GUVs) and supported
phospholipid bilayers (SPBs), were detd. by fluorescence correlation
spectroscopy (FCS) using the Z-scan approach. For the first time, the performed
measurements on 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) bilayers
maintain exactly the same exptl. conditions for both systems, which allows for
a quant. comparison of lipid diffusion in these two commonly used model
membranes. The results obtained revealed that the lipid mobility in
free-standing bilayers (D = 7.8+-0.8 mm2 s-1) is significantly higher than in
the bilayer created on the solid support (mica) (D = 3.1+-0.3 mm2 s-1).
Quinlan, R. J. and G. D. Reinhart.
(2006) Effects of Protein-Ligand Associations on the Subunit Interactions of
Phosphofructokinase from B. stearothermophilus. Biochemistry 45(38):11333-11341.
Differences
between the crystal structures of inhibitor-bound and uninhibited forms of
phosphofructokinase (PFK) from B. stearothermophilus have led to a structural
model for allosteric inhibition by phosphoenolpyruvate (PEP) wherein a
dimer-dimer interface within the tetrameric enzyme undergoes a quaternary
shift. We have developed a labeling and hybridization technique to generate a
tetramer with subunits simultaneously contg. two different extrinsic
fluorophores in known subunit orientations. This construct has been utilized in
the examn. of the effects of allosteric ligand and substrate binding on the
subunit affinities of tetrameric PFK using several biophys. and spectroscopic
techniques including 2-photon, dual-channel fluorescence correlation
spectroscopy (FCS). We demonstrate that PEP-binding at the allosteric site is
sufficient to reduce the affinity of the active site interface from beyond the
limits of exptl. detection to nanomolar affinity, while conversely
strengthening the interface at which it is bound. The reduced interface
affinity is specific to inhibitor binding because binding the activator ADP at
the same allosteric site causes no redn. in subunit affinity. With inhibitor
bound, the weakened subunit affinity has allowed the kinetics of dimer assocn.
to be elucidated.
Rao, R., R. Langoju, M. Goesch, P.
Rigler, A. Serov and T. Lasser. (2006) Stochastic Approach to Data Analysis
in Fluorescence Correlation Spectroscopy. Journal of Physical Chemistry A 110(37):10674-10682.
Fluorescence
correlation spectroscopy (FCS) has emerged as a powerful technique for
measuring low concns. of fluorescent mols. and their diffusion consts. In FCS,
the exptl. data is conventionally fit using std. local search techniques, for
example, the Marquardt-Levenberg (ML) algorithm. A prerequisite for these
categories of algorithms is the sound knowledge of the behavior of fit
parameters and in most cases good initial guesses for accurate fitting, otherwise
leading to fitting artifacts. For known fit models and with user experience
about the behavior of fit parameters, these local search algorithms work
extremely well. However, for heterogeneous systems or where automated data
anal. is a prerequisite, there is a need to apply a procedure, which treats FCS
data fitting as a black box and generates reliable fit parameters with accuracy
for the chosen model in hand. A computational approach is presented to analyze
FCS data by means of a stochastic algorithm for global search called PGSL, an
acronym for probabilistic global search Lausanne. This algorithm does not
require any initial guesses and does the fitting in terms of searching for
solns. by global sampling. It is flexible as well as computationally faster at
the same time for multiparameter evaluations. The performance study is
presented of PGSL for two-component with triplet fits. The statistical study
and the goodness of fit criterion for PGSL are also presented. The robustness
of PGSL on noisy exptl. data for parameter estn. is also verified. The scope of
PGSL is extended by a hybrid anal. wherein the output of PGSL is fed as initial
guesses to ML. Reliability studies show that PGSL and the hybrid combination of
both perform better than ML for various thresholds of the mean-squared error
(MSE).
Remaut, K., B. Lucas, K. Braeckmans,
N. N. Sanders, J. Demeester and S. C. De Smedt. (2006) Delivery of
Phosphodiester Oligonucleotides: Can DOTAP/DOPE Liposomes Do the Trick?
Biochemistry 45(6):1755-1764.
Delivering
phosphodiester ONs (PO-ONs) remains an attractive but challenging goal in
antisense therapy. Both in the literature and in our expts., most cationic
liposomes fail in generating an antisense effect with PO-ONs, while they
succeed with chem. modified ONs such as phosphothioate ONs (PS-ONs). This work
aims to explain the biol. activity of PO- and PS-ONs delivered by DOTAP/DOPE
liposomes based on a detailed understanding of their cell biol. behavior by
means of fluorescence correlation spectroscopy and confocal laser scanning
microscopy. We conclude that DOTAP/DOPE liposomes are not suited to deliver
PO-ONs due to the release of naked PO-ONs in the cytosol at the time of the
endosomal escape of the liposomes and the subsequent rapid degrdn. of the naked
PO-ONs. Carriers that would not release the PO-ONs upon endosomal escape but
would continue to carry the PO-ONs until they arrive at the target mRNA could
therefore be better suited to delivering PO-ONs. In the case of PS-ONs, the ONs
are not degraded upon release at the time of the endosomal escape of the
liposomes, creating a pool of intact, biol. active PS-ONs and thus making
DOTAP/DOPE liposomes mainly suitable for delivering nuclease resistant ONs.
However, the cells seemed to display an export pathway for removing intact
PS-ONs from the cells, limiting the presence of naked PS-ONs in the nucleus to
.apprx.8 h following the delivery.
Rhoades, E., T. F. Ramlall, W. W.
Webb and D. Eliezer. (2006) Quantification of a-synuclein binding to lipid
vesicles using fluorescence correlation spectroscopy. Biophysical Journal 90(12):4692-4700.
a-Synuclein
(aS) is a sol. synaptic protein that is the major proteinaceous component of
insol. fibrillar Lewy body deposits that are the hallmark of Parkinson's disease.
The interaction of aS with synaptic vesicles is thought to be crit. both to its
normal function as well as to its pathol. role in Parkinson's disease. We
demonstrate the use of fluorescence correlation spectroscopy as a tool for
rapid and quant. anal. of the binding of aS to large unilamellar vesicles of
various lipid compns. We find that aS binds preferentially to vesicles contg.
acidic lipids, and that this interaction can be blocked by increasing the
concn. of NaCl in soln. Neg. charge is not the only factor detg. binding, as we
clearly observe binding to vesicles composed entirely of zwitterionic lipids.
Addnl., we find enhanced binding to lipids with less bulky headgroups.
Quantification of the protein-to-lipid ratio required for binding to different
lipid compns., combined with other data in the literature, yields an upper
bound est. for the no. of lipid mols. required to bind each individual mol. of
aS. Our results demonstrate that fluorescence correlation spectroscopy provides
a powerful tool for the quant. characterization of aS-lipid interactions.
Ries, J. and P. Schwille. (2006) Studying
slow membrane dynamics with continuous wave scanning fluorescence correlation
spectroscopy. Biophysical Journal 91(5):1915-1924.
Here
the authors discuss the application of scanning fluorescence correlation
spectroscopy (SFCS) using continuous wave excitation to analyze membrane
dynamics. The high count rate per mol. enables the study of very slow diffusion
in model and cell membranes, as well as the application of two-foci
fluorescence cross-correlation spectroscopy for parameter-free detn. of
diffusion consts. The combination with dual-color fluorescence
cross-correlation spectroscopy with continuous or pulsed interleaved excitation
allows binding studies on membranes. Redn. of photobleaching, higher
reproducibility, and stability compared to traditional FCS on membranes, and
the simple implementation in a com. microscopy setup make SFCS a valuable addn.
to the pool of fluorescence fluctuation techniques.
Rigler, P. and W. Meier. (2006) Encapsulation
of fluorescent molecules by functionalized polymeric nanocontainers:
investigation by confocal fluorescence imaging and fluorescence correlation
spectroscopy. Journal of the American Chemical Society 128(1):367-373.
Nanocontainers
(NCs) were prepd. from amphiphilic triblock copolymers, having an av. mol. wt.
of around 8000 g/mol, by using previously published prepn. methods consisting
of dispersing the polymer in an aq. buffer soln. contg. mols. for encapsulation.
A small mol. wt. fluorophore, sulforhodamine B, as well as the fluorescent
protein avidin labeled with Alexa 488 were encapsulated, and the resulting
nanocontainers were characterized using fluorescence correlation spectroscopy
(FCS) and fluorescence cross-correlation spectroscopy (FCCS). Nanocontainer
size detn. by FCS is very robust and compares well with results obtained from
photon correlation spectroscopy: the measured diams. of the polymeric
nanocontainers vary between 140 and 172 nm. Encapsulation of fluorescent mols.
was detd. by evaluating the mol. brightness of nanocontainers with an
encapsulated fluorescently labeled protein (avidin-Alexa 488). Results
indicated that the no. of encapsulated avidin-Alexa 488 mols. corresponds well
with the initial concn. of the fluorescently labeled protein and the
encapsulated vol. A nanocontainer binding assay was developed using
biotinylated fluorescently labeled nanocontainers. Binding of biotinylated
nanocontainers to fluorescently labeled streptavidin was followed by
fluorescence cross-correlation spectroscopy. The intrinsic dissocn. const., Kd,
of labeled streptavidin to the ligand-modified nanocontainers is 1.7 +- 0.4 *
10-8 M, and about 1921 +- 357 mols. of labeled streptavidin are bound to each
nanocontainer.
Ruetinger, S., R. Macdonald, B.
Kramer, F. Koberling, M. Roos and E. Hildt. (2006) Accurate single-pair
Forster resonant energy transfer through combination of pulsed interleaved
excitation, time correlated single-photon counting, and fluorescence
correlation spectroscopy. Journal of Biomedical Optics 11(2):024012/1-024012/9.
Quant.
distance measurements are difficult to obtain in spite of the strong distance
dependency of the energy transfer efficiency. One problem for the
interpretation of the Forster resonant energy transfer (FRET) efficiency is the
so-called zero-efficiency peak caused by FRET pairs with missing or
nonfluorescent acceptors. Other problems occurring are direct excitation of the
acceptor, spectral crosstalk, and the detn. of the quantum efficiency of the
dyes as well as the detector sensitivity. Our approach to overcome these
limitations is based on the pulsed-interleaved excitation (PIE) of both the
acceptor and the donor mol. PIE is used to excite the acceptor dye independently
of the FRET process and to prove its existence via fluorescence. This technique
enables us to differentiate a FRET mol., even with a very low FRET efficiency,
from a mol. with an absent or non-fluorescent acceptor. Crosstalk, direct
acceptor excitation, and mol. brightness of acceptor and donor mols. are detd.
by analyzing the data with fluorescence correlation spectroscopy (FCS). FRET
efficiencies of the same data set are also detd. by analyzing the lifetimes of
the donor fluorophores. The advantages of the PIE-FRET approach are
demonstrated on a polyproline assay labeled with Alexa-555 and Alexa-647 as
donor and acceptor, resp.
Samiee, K. T., J. M. Moran-Mirabal,
Y. K. Cheung and H. G. Craighead. (2006) Zero mode waveguides for
single-molecule spectroscopy on lipid membranes. Biophysical Journal 90(9):3288-3299.
Zero
mode waveguides (ZMWs), subwavelength optical nanostructures with dimensions
ranging from 50 to 200 nm, have been used to study systems involving
ligand-receptor interactions. The authors show that under proper conditions,
lipid membranes will invaginate into the nanostructures, which confine optical
excitation to subattoliter vols. Fluorescence correlation spectroscopy (FCS)
was used to characterize the diffusion of fluorescently tagged lipids in
liq.-disordered phase 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC)
and gel phase 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) membranes
incubated on the nanostructured surface. In contrast to the POPC, DSPC
membranes did not appear to enter the structures, suggesting that invagination
is dependent on membrane rigidity. Although correlation curves obtained from
POPC membranes conformed to previously derived models for diffusion in the
evanescent field within the nanostructure, the diffusion consts. obtained were
systematically lower than expected. The validity of the one-dimensional
diffusion model for membrane diffusion is discussed and it is concluded that
the erroneous diffusion consts. are a result of nontrivial membrane conformation
within the ZMWs. Addnl., FCS was used to characterize the fraction of
fluorescently labeled tetanus toxin C fragment bound to a ganglioside-populated
POPC membrane within the ZMWs. This allowed the detn. of the toxin's equil.
binding const. at a concn. of 500 nM; higher than possible with
diffraction-limited FCS. To the authors' knowledge, the results presented here
are the first reported for supported lipid bilayers in nanostructured devices.
Furthermore, they open the possibility of studying membrane imbedded receptors
and proteins at physiol. concns. with single-mol. resoln.
Schaefer, I. B., S. M. Bailer, M. G.
Dueser, M. Boersch, R. A. Bernal, D. Stock and G. Grueber. (2006) Crystal
Structure of the Archaeal A1AO ATP Synthase Subunit B from Methanosarcina mazei
Go1: Implications of Nucleotide-binding Differences in the Major A1AO Subunits
A and B. Journal of Molecular Biology 358(3):725-740.
The
A1AO ATP synthase from archaea represents a class of chimeric
ATPases/synthases, whose function and general structural design share
characteristics both with vacuolar V1VO ATPases and with F1FO ATP synthases.
The primary sequences of the two large polypeptides A and B, from the catalytic
part, are closely related to the eukaryotic V1VO ATPases. The chimeric nature
of the A1AO ATP synthase from the archaeon Methanosarcina mazei Go1 was
investigated in terms of nucleotide interaction. Here, we demonstrate the
ability of the overexpressed A and B subunits to bind ADP and ATP by
photoaffinity labeling. Matrix-assisted laser desorption/ionization
time-of-flight mass spectrometry was used to map the peptide of subunit B
involved in nucleotide interaction. Nucleotide affinities in both subunits were
detd. by fluorescence correlation spectroscopy, indicating a weaker binding of
nucleotide analogs to subunit B than to A. In addn., the nucleotide-free
crystal structure of subunit B is presented at 1.5 .ANG. resoln., providing the
first view of the so-called non-catalytic subunit of the A1AO ATP synthase.
Superposition of the A-ATP synthase non-catalytic B subunit and the F-ATP
synthase non-catalytic a subunit provides new insights into the similarities
and differences of these nucleotide-binding ATPase subunits in particular, and
into nucleotide binding in general. The arrangement of subunit B within the
intact A1AO ATP synthase is presented.
Schlagberger, X., J. Bayer, J. O.
Raedler and R. R. Netz. (2006) Diffusion of a single semiflexible charged
polymer. Europhysics Letters 76(2):346-352.
The
diffusion of charged semiflexible polymers is studied by hydrodynamic
simulations. The salt-dependent diffusion const. exhibits a shallow min. at a
screening length comparable to the polymer length. Fluorescence correlation
spectroscopy data for 394 bp ds-DNA fragments confirm the relative
insensitivity of diffusion over a wide range of salt concns. A scaling
expression for the diffusion const. of a neutral semiflexible chain
encompassing the rod, the intermediate ideal and the asymptotic swollen regime
is constructed.
Sherman, E. and G. Haran. (2006) Coil-globule
transition in the denatured state of a small protein. Proceedings of the
National Academy of Sciences of the United States of America 103(31):11539-11543.
Upon
transfer from strongly denaturing to native conditions, proteins undergo a
collapse that either precedes folding or occurs simultaneously with it. This
collapse is similar to the well known coil-globule transition of polymers.
Here, the authors employed single-mol. fluorescence methods to fully
characterize the equil. coil-globule transition in the denatured state of the
64-amino-acid IgG-binding domain of protein L. By using single-mol. FRET
measurements on freely diffusing individual mols., the authors detd. the radius
of gyration of the protein, which showed a gradual expansion as the concn. of
the denaturant, guanidine-HCl, was increased all the way up to 7M. This
expansion was obsd. also in fluorescence correlation spectroscopy measurements
of the hydrodynamic radius of the protein. The authors analyzed the radius of
gyration measurements using the theory of the coil-globule transition of I. C.
Sanchez (1979), which balances the excluded vol. entropy of the chain with the
av. inter-residue interaction energy. In particular, the authors calcd. the
solvation energy of the denatured protein, a property that was not readily
accessible in other expts. The dependence of this energy on denaturant concn.
was nonlinear, contrasting with the common linear extrapolation method used to
describe denaturation energy. Interestingly, a fit to the binding model of
chem. denaturation suggested a single denaturant binding site per protein
residue. The size of the denatured protein under native conditions could be
extrapolated from the data as well, showing that the fully collapsed state of
protein was only .apprx.10% larger than the folded state.
Siegberg, D., C. M. Roth and D.-P.
Herten. (2006) Single molecule fluorescence spectroscopy: approaches toward
quantitative investigations of structure and dynamics in living cells.
Proceedings of SPIE-The International Society for Optical Engineering 6092(Ultrasensitive
and Single-Molecule Detection Technologies):609202/1-609202/5.
The
investigation of the structure and dynamics of biomols. and biomol. assemblies
in living cells is of current interest in mol. biol. Recent developments in
single mol. fluorescence spectroscopy (SMFS) have opened ways for investigating
the dynamics and stoichiometry of individual biomol. complexes e.g., by
application of single pair fluorescence resonance energy transfer (spFRET) with
alternating laser excitation (ALEX), and by improved labels and labeling
techniques. In the recent years, we have developed a set of techniques that
allow the detn. of the spatial distribution of single fluorescent mols. and
their identification by spectrally-resolved fluorescence lifetime imaging
microscopy (SFLIM) as well as the observation of the dynamics of individual
mols. immobilized on surfaces. Based on SFLIM we currently focus on
investigating the diffusion kinetics of biomols. in living cells. By combining
high-resoln. confocal fluorescence microscopy of single mols. with fluorescence
correlation spectroscopy (FCS) we seek to quantitate diffusion coeffs. and
concns. of relevant fluorescently labeled biomols. within living cells thereby
visualizing the heterogeneous distribution of local mobilities in the sample.
The simultaneously acquired fluorescence intensity and lifetime images can
further be used for addnl. single point measurements for obtaining i.e.,
information about the stoichiometry of immobilized biomol. complexes based on
photon anti-bunching. In addn. the simultaneous acquisition of multiple
characteristic properties by SFLIM, like spectral emission bands and
fluorescence lifetime, offers the opportunity to discriminate different
fluorescent probes and autofluorescence.
Sisan, D. R., R. Arevalo, C. Graves,
R. McAllister and J. S. Urbach. (2006) Spatially resolved fluorescence
correlation spectroscopy using a spinning disk confocal microscope.
Biophysical Journal 91(11):4241-4252.
We
develop an extension of fluorescence correlation spectroscopy (FCS) using a
spinning disk confocal microscope. This approach can spatially map diffusion
coeffs. or flow velocities at up to .apprx.105 independent locations
simultaneously. Com. available cameras with frame rates of 1000 Hz allow FCS
measurements of systems with diffusion coeffs. D.apprx.10-7 cm2/s or smaller.
This speed is adequate to measure small microspheres (200-nm diam.) diffusing
in water, or hindered diffusion of macromols. in complex media (e.g., tumors,
cell nuclei, or the extracellular matrix). There have been a no. of recent
extensions to FCS based on laser scanning microscopy. Spinning disk confocal
microscopy, however, has the potential for significantly higher speed at high
spatial resoln. We show how to account for a pixel size effect encountered with
spinning disk confocal FCS that is not present in std. or scanning FCS, and we
introduce a new method to correct for photobleaching. Finally, we apply
spinning disk confocal FCS to microspheres diffusing in Type I collagen, which
show complex spatially varying diffusion caused by hydrodynamic and steric
interactions with the collagen matrix.
Sonehara, T., T. Anazawa and K.
Uchida. (2006) Improvement of Biomolecule Quantification Precision and Use
of a Single-Element Aspheric Objective Lens in Fluorescence Correlation
Spectroscopy. Analytical Chemistry 78(24):8395-8405.
We
found a way to increase the precision with which biomols. present at concns.
below 10-10 M can be quantified by fluorescence correlation spectroscopy (FCS).
The effectiveness of the way was demonstrated exptl. by using a single-element
aspheric objective lens, which was newly developed to reduce the cost of FCS instruments.
In the first part of this paper, the relative std. deviation (RSD) of FCS-based
concn. measurements is estd. theor. by an anal. approxn. assuming the detection
vol. profiles in FCS setups to be Gaussian and by mol. simulations in which
more realistic profiles are calcd. from phys. parameters of the measurement
setups. In a limit of infinitely bright mols. and zero background emission, the
anal. approxn. predicts that the RSD at a concn. is minimized when the mean no.
of mols. in a detection vol. is .apprx.0.5. A detection vol. of the order of
10-13 L thus gives smaller RSD values for concns. from 10-11 to 10-10 M than
does one of the order of 10-15 L, which is widely used in FCS. This prediction
is supported by the mol. simulations, taking into account the finite mol.
brightness and background emission. In the second part of the paper, the RSD is
evaluated exptl. with an FCS setup with a detection vol. of 1.1*10-13 L. The
newly developed objective lens, serving as the bottom of the sample cell in
this setup, has a large numerical aperture (0.9) without using immersion liq.
When a calibration line was made by 30-s FCS measurements of Cy3-labeled,
112-mer single-stranded DNA solns., the RSD roughly agreed with the simulation
result and was less than 0.1 for DNA concns. from 2*10-11 to 10-10 M.
Szymanski, J., A. Patkowski, J.
Gapinski, A. Wilk and R. Holyst. (2006) Movement of Proteins in an
Environment Crowded by Surfactant Micelles: Anomalous versus Normal Diffusion.
Journal of Physical Chemistry B 110(14):7367-7373.
Small
proteins move in crowded cell compartments by anomalous diffusion. In many of
them, e.g., the endoplasmic reticulum, the proteins move between lipid
membranes in the aq. lumen. Mol. crowding in vitro offers a systematic way to study
anomalous and normal diffusion in a well controlled environment not accessible
in vivo. We prepd. a crowded environment in vitro consisting of hexaethylene
glycol monododecyl ether (C12E6) nonionic surfactant and water and obsd.
lysozyme diffusion between elongated micelles. We have fitted the data obtained
in fluorescence correlation spectroscopy using an anomalous diffusion model and
a two-component normal diffusion model. For a small concn. of surfactant (below
4 wt. %) the data can be fitted by single-component normal diffusion. For
larger concns. the normal diffusion fit gave two components: one very slow and
one fast. The amplitude of the slow component grows with C12E6 concn. The ratio
of diffusion coeffs. (slow to fast) is on the order of 0.1 for all concns. of
surfactant in the soln. The fast diffusion is due to free proteins while the
slow one is due to the protein-micelle complexes. The protein-micelle
interaction is weak since even in a highly concd. soln. (35% of C12E6) the
amplitude of the slow mode is only 10%, despite the fact that the av. distance
between the micelles is the same as the size of the protein. The anomalous
diffusion model gave the anomality index ({r2(t)} .apprx. ta), a monotonically
decreasing from a = 1 (at 4% surfactant) to a = 0.88 (at 37% surfactant). The
fits for two-component normal diffusion and anomalous diffusion were of equally
good quality, but the phys. interpretation was only straightforward for the
former.
Szymanski, J., A. Patkowski, A.
Wilk, P. Garstecki and R. Holyst. (2006) Diffusion and Viscosity in a
Crowded Environment: from Nano- to Macroscale. Journal of Physical
Chemistry B 110(51):25593-25597.
Although
water is the chief component of living cells, food, and personal care products,
the supramol. components make their viscosity larger than that of water by
several orders of magnitude. Using fluorescence correlation spectroscopy (FCS),
photon correlation spectroscopy (PCS), NMR, and rheol. data, we show how the
viscosity changes from the value for water at the mol. scale to the large
macroviscosity. We detd. the viscosity experienced by nanoprobes (of sizes from
0.28 to 190 nm) in aq. micellar soln. of hexaethylene-glycol-monododecyl-ether
(in a range of concn. from 0.1% wt./wt. to 35% wt./wt.) and identified a clear
crossover at the length scale of 17 +- 2 nm (slightly larger than persistence
length of micelles) at which viscosity acquires its macroscopic value. The
sharp dependence of the viscosity coeffs. on the size of the probe in the
nanoregime has important consequences for diffusion-limited reactions in
crowded environments (e.g., living cells).
Tabouillot, T., R. Gullapalli and P.
J. Butler. (2006) Monitoring cellular mechanosensing using time-correlated
single photon counting. Proceedings of SPIE-The International Society for
Optical Engineering 6372(Advanced Photon Counting
Techniques):63720D/1-63720D/8.
Endothelial
cells (ECs) convert mech. stimuli into chem. signaling pathways to regulate
their functions and properties. It is hypothesized that perturbation of
cellular structures by force is accompanied by changes in mol. dynamics. In
order to address these fundamental issues in mechanosensation and transduction,
we have developed a hybrid multimodal microscopy - time-correlated single
photon counting (TCSPC) spectroscopy system intended to det. time- and position
dependent mech.-induced changes in the dynamics of mols. in live cells as detd.
from fluorescence lifetimes and autocorrelation anal. (fluorescence correlation
spectroscopy). Colocalization of cell-structures and mech.-induced changes in
mol. dynamics can be done in post-processing by comparing TCSPC data with 3-D
models generated from total internal reflection fluorescence (TIRF),
differential interference contrast (DIC), epifluorescence, and deconvolution.
We present control expts. in which the precise location of the apical cell
membrane with respect to a confocal probe is assessed using information
obtainable only from TCSPC. Such positional accuracy of TCSPC measurements is
essential to understanding the role of the membrane in mechanotransduction. We
predict that TCSPC will become a useful method to obtain high temporal and
spatial resoln. information on localized mech. phenomena in living endothelial
cells. Such insight into mechanotransduction phenomenon may uncover the origins
of mech.-related diseases such as atherosclerosis.
Tetin, S. Y., Q. Ruan, S. C.
Saldana, M. R. Pope, Y. Chen, H. Wu, M. S. Pinkus, J. Jiang and P. L.
Richardson. (2006) Interactions of Two Monoclonal Antibodies with BNP: High
Resolution Epitope Mapping Using Fluorescence Correlation Spectroscopy.
Biochemistry 45(47):14155-14165.
Structure-function
studies of antibody-antigen systems include the identification of amino acid
residues in the antigen that interact with an antibody and elucidation of their
individual contributions to binding affinity. We used fluorescence correlation
spectroscopy (FCS) and alanine-scanning mutagenesis to characterize the
interactions of brain natriuretic peptide (BNP) with two monoclonal antibodies.
Human BNP is a 32 amino acid residue long cyclic polypeptide with the ring
structure confined between cysteines in positions 10 and 26. It is an important
cardiovascular hormone and a valuable diagnostic cardiac marker. We compare the
binding strength of the N-terminus Alexa488-labeled BNP, native cyclic BNP, BNP
alanine-substituted mutants, linear BNP, and its short fragments to det. the
individual contributions of amino acid residues included in the continuous antigenic
epitopes that are recognized by two different monoclonal antibodies raised
toward BNP. Implementation of FCS for these studies offers all of the
advantages of soln. phase measurements, including high sensitivity, simplicity
of manipulation with reagents, and elimination of solid phase interferences or
sepn. steps. Significant differences in the mol. masses of the free and
antibody bound BNP results in a substantial (.apprx.2.5-times) increase in the
diffusion rates. Detn. of the binding consts. and inhibition effects by
measuring the diffusion rates of the ligand at the single mol. level introduces
the ultimate opportunity for researching systems where the fluorescence
intensity and/or fluorescence anisotropy do not change upon interaction of the
ligand with the protein. Monoclonal antibodies 106.3 and BC203 demonstrate high
affinities to BNP and bind two distant epitopes forming robust antibody
sandwiches. Both antibodies are used in Abbott diagnostic assays on AxSYM, IMx,
and Architect platforms.
Tilz, G. P., M. Wiltgen, U. Demel,
C. Faschinger, H. Schmiedinger and A. Hermetter. (2006) Introduction of
three complementary technologies into clinical medicine: Developmemt of new
diagnostic and prognostic parameters. American Journal of Immunology 2(1):12-18.
Mol.
medicine leads us to understand some diseases at the mol. level. Examples are
the anal. of immune complexes and receptor - anti receptor compds. used in
clin. medicine. Structural changes of some serum proteins occur in
inflammation, neoplasie and autoimmunity. The detection and anal. of such
structural modifications may offer a new field for the diagnosis, prognosis and
therapy of some diseases. Modern medicine requires new technologies with high
sensitivity, specificity and applicability. For the first time in Austria we
have combined fluorescence correlation spectroscope (FCS), Surface enhanced
laser desorption ionisation - time of flight (SELDI-TOF) and the mol. modeling
and visualization system according to the computer enhanced programs. Exptl.
and computational methods are combined in such a way, that clin. data can be
interpreted by theor. methods at a mol. level or vice versa the computational
output delivers input for new investigations. We will test our combination of
exptl. and theor. methods on well suited media such as: aq. humor, sera and
highly purified biologicals. These media contain albumin and other substances
which might be modified structurally by tumor enzymes and inflammatory
products, hence giving us a valuable indication of the underlying diseases. All
the new methods quoted have their disadvantages which can be bypassed by
multiplexing, what we have done. So we came to the detection of albumin in the
aq. humor and an increase of sensitivity for the detection of the Goodpasture-antigen.
Results from structural anal. of albumin and collagen IV under normal
conditions will serve as refs. for further investigations. Software for the
structural anal. has been developed by us and results will be presented at this
place. One method brings us single results. In view of the spectrum of
parameters relevant to clin. entities, multiplexing is a new way of
development. Since the technologies are new, the scientifically interested
reader should be informed about the matters arising.
Tsay, J. M., S. Doose and S. Weiss.
(2006) Rotational and Translational Diffusion of Peptide-Coated CdSe/CdS/ZnS
Nanorods Studied by Fluorescence Correlation Spectroscopy. Journal of the
American Chemical Society 128(5):1639-1647.
CdSe/CdS/ZnS
nanorods (NRs) of three aspect ratios were coated with phytochelatin-related
peptides and studied using fluorescence correlation spectroscopy (FCS). Theor.
predictions of the NRs' rotational diffusion contribution to the correlation
curves were exptl. confirmed. We monitored rotational and translational
diffusion of NRs and extd. hydrodynamic radii from the extd. diffusion consts.
Translational and rotational diffusion consts. (Dtrans and Drot) for NRs were
in good agreement with Tirado and Garcia de la Torre's as well as with
Broersma's theories when accounting for the ligand dimensions. NRs fall in the
size range where rotational diffusion can be monitored with higher sensitivity
than translational diffusion due to a steeper length dependence, Drot .apprx.
L-3 vs. Dtrans .apprx. L-1. By titrating peptide-coated NRs with bovine serum
albumin, we monitored (nonspecific) binding through rotational diffusion and
showed that Drot is an advantageous observable for monitoring binding.
Monitoring rotational diffusion of bioconjugated NRs using FCS might prove to
be useful for observing binding and conformational dynamics in biol. systems.
Veldhuis, G., M. Hink, V. Krasnikov,
G. Van Den Bogaart, J. Hoeboer, A. J. W. G. Visser, J. Broos and B. Poolman.
(2006) The oligomeric state and stability of the mannitol transporter,
EnzymeIImtl, from Escherichia coli: a fluorescence correlation spectroscopy
study. Protein Science 15(8):1977-1986.
Numerous
membrane proteins function as oligomers both at the structural and functional
levels. The mannitol transporter from Escherichia coli, EnzymeIImtl, is a
member of the phosphoenolpyruvate-dependent phosphotransferase system. During
the transport cycle, mannitol is phosphorylated and released into the cytoplasm
as mannitol-1-phosphate. Several studies have shown that EIImtl functions as an
oligomeric species. However, the oligomerization no. and stability of the
oligomeric complex during different steps of the catalytic cycle, e.g.,
substrate binding and/or phosphorylation of the carrier, is still under
discussion. In this paper, we have addressed the oligomeric state and stability
of EIImtl using fluorescence correlation spectroscopy. A functional
double-cysteine mutant was site-specifically labeled with either Alexa Fluor
488 or Alexa Fluor 633. The subunit exchange of these two batches of proteins
was followed in time during different steps of the catalytic cycle. The most
important conclusions are that (1) in a detergent-solubilized state, EIImtl is
functional as a very stable dimer; (2) the stability of the complex can be
manipulated by changing the intermicellar attractive forces between PEG-based
detergent micelles; (3) substrate binding destabilizes the complex, whereas
phosphorylation increases the stability; and (4) substrate binding to the
phosphorylated species partly antagonizes the stabilizing effect.
Wang, Z., J. V. Shah, M. W. Berns
and D. W. Cleveland. (2006) In vivo quantitative studies of dynamic
intracellular processes using fluorescence correlation spectroscopy.
Biophysical Journal 91(1):343-351.
It
has been a significant challenge to quant. study the dynamic intracellular
processes in live cells. These studies are essential for a thorough
understanding of the underlying mechanisms regulating the signaling pathways
and the transitions between cell cycle stages. The authors' studies of Cdc20,
an important mitotic checkpoint protein, throughout the cell cycle demonstrate
that fluorescence correlation spectroscopy is a powerful tool for in vivo
quant. studies of dynamic intracellular processes. In this study, Cdc20 is
present primarily in a large complex (>1 Mda) during interphase with a
diffusion const. of 1.8+-0.1 mm2/s and a concn. of 76+-24 nM, consistent with
its assocn. with the APC/C. During mitosis, however, a proportion of Cdc20
dissocs. from APC/C at a rate of 12 pM/s into a sol. pool with a diffusion
const. of 19.5+-5.0 mm2/s, whose size is most consistent with free Cdc20. This
free pool accumulates to 50% of total Cdc20 (.apprx.40 nM) during chronic
activation of the mitotic checkpoint but disappears during mitotic exit at a
rate of 31 pM/s. The obsd. changes in the biochem. assembly states of Cdc20
closely correlate to the known temporal pattern of the activity of APC/CCdc20
in mitosis. Photon counting histograms reveal that both complexes contain only
a single mol. of Cdc20. The underlying mechanisms of the activities of
APC/CCdc20 throughout the cell cycle are discussed in light of the authors'
exptl. observations.
Weisshart, K. (2006) The LSM 5
family: an integrated imaging and spectroscopic platform for the study of
cellular dynamic processes. Proceedings of SPIE-The International Society
for Optical Engineering 6089(Multiphoton Microscopy in the Biomedical
Sciences VI):608909/1-608909/7.
The
elucidation of diffusion processes and mol. interactions and their relation to
compartments and structures will be essential to understand cellular functions
in detail. Often it is not the av. signal that is of interest but the behavior
of single mols. which behave as individuals. Fluorescence based assays have
revolutionized the way we can observe mols. at work in their natural cellular
settings and they have now also become available for single mol. studies. These
technologies comprise Fluorescence Fluctuation Anal. (FFA) including
Fluorescence Correlation Spectroscopy (FCS), Fluorescence Redistribution After
Photobleaching (FRAP), Foerster Resonance Energy Transfer (FRET) and
Fluorescence Lifetime Imaging (FLIM). Esp. for dual color expts. and when
dealing with delicate samples the employment of multiphoton microscopy using
the aforementioned technologies can be of great benefit. Ideal instruments to
study single mols. would therefore need to accommodate equipment that allow for
fast time resoln., adequate detectors and lasers as well as integrated work
flows. In this contribution we discuss the newest developments in com.
instrumentation and software at Carl Zeiss towards highly sensitive imaging in
combination with spectroscopic anal.
Wenger, J., H. Rigneault, J. Dintinger,
D. Marguet and P.-F. Lenne. (2006) Single-Fluorophore Diffusion in a Lipid
Membrane over a Subwavelength Aperture. Journal of Biological Physics 32(1):SN1-SN4.
The
authors use submicrometer apertures milled in an aluminum film to study the diffusion
dynamics of b-Bodipy-FL-C5-HPC (Bodipy-PC) fluorophores in a lipid
dioleoylphosphatidylcholine (DOPC) multilayer. The observation vol. is limited
by the aperture diam., well below the optical wavelength. This spatial resoln.
improvement comes together with an enhancement of the detected fluorescence per
mol. as compared to an open sample, with a significant increase up to 3.5
times.
Werner, J. H., R. Joggerst, R. B.
Dyer and P. M. Goodwin. (2006) A two-dimensional view of the folding energy
landscape of cytochrome c. Proceedings of the National Academy of Sciences
of the United States of America 103(30):11130-11135.
Time-correlated
single photon counting (TCSPC) was combined with fluorescence correlation
spectroscopy (FCS) to study the transition between acid-denatured states and
the native structure of cytochrome c (Cyt c) from Saccharomyces cerevisiae. The
use of these techniques in concert proved to be more powerful than either
alone, yielding a two-dimensional picture of the folding energy landscape of
Cyt c. TCSPC measured the distribution of distances between the heme of the
protein and a covalently attached dye mol. at residue C102 (one folding
reaction coordinate), whereas FCS measured the hydrodynamic radius (a second
folding reaction coordinate) of the protein over a range of pH values. These
two independent measurements provide complementary information regarding
protein conformation. We see evidence for a well defined folding intermediate
in the acid renaturation folding pathway of this protein reflected in the
distribution of lifetimes needed to fit the TCSPC data. Moreover, FCS studies
revealed this intermediate state to be in dynamic equil. with unfolded
structures, with conformational fluctuations into and out of this intermediate
state occurring on an ~30-ms time scale.
Westphal, A. H., A. Matorin, M. A.
Hink, J. W. Borst, W. J. H. Van Berkel and A. J. W. G. Visser. (2006) Real-time
Enzyme Dynamics Illustrated with Fluorescence Spectroscopy of p-Hydroxybenzoate
Hydroxylase. Journal of Biological Chemistry 281(16):11074-11081.
We
have used the flavoenzyme p-hydroxybenzoate hydroxylase (PHBH) to illustrate
that a strongly fluorescent donor label can communicate with the flavin via
single-pair Foerster resonance energy transfer (spFRET). The accessible Cys-116
of PHBH was labeled with two different fluorescent maleimides with full
preservation of enzymic activity. One of these labels shows overlap between its
fluorescence spectrum and the absorption spectrum of the FAD prosthetic group
in the oxidized state, while the other fluorescent probe does not have this
spectral overlap. The spectral overlap strongly diminished when the flavin
becomes reduced during catalysis. The donor fluorescence properties can then be
used as a sensitive antenna for the flavin redox state. Time-resolved
fluorescence expts. on ensembles of labeled PHBH mols. were carried out in the
absence and presence of enzymic turnover. Distinct changes in fluorescence
decays of spFRET-active PHBH can be obsd. when the enzyme is performing
catalysis using both substrates p-hydroxybenzoate and NADPH. Single-mol.
fluorescence correlation spectroscopy on spFRET-active PHBH showed the presence
of a relaxation process (relaxation time of 23 ms) that is related to
catalysis. In addn., in both labeled PHBH prepns. the no. of enzyme mols.
reversibly increased during enzymic turnover, indicating that the dimer-monomer
equil. is affected.
Winkler, R. G., S. Keller and J. O.
Radler. (2006) Intramolecular dynamics of linear macromolecules by
fluorescence correlation spectroscopy. Physical Review E: Statistical,
Nonlinear, and Soft Matter Physics 73(4-1):041919/1-041919/14.
A
theor. description of the dynamics of DNA mols. and actin filaments in soln. as
measured exptl. by fluorescence correlation spectroscopy is provided and
compared to recent exptl. results. Particular attention is paid to the
contribution of the intramol. dynamics to the fluorescence correlation
function. Using a semiflexible chain model, a theor. expression is presented
for the fluorescence correlation spectroscopy correlation function. The
dependence of this function on various model parameters, such as chain length,
persistence length, and fluorescence label d., is discussed. Our investigations
show that the intramol. dynamics provides a significant contribution or even
dominates the correlation function as soon as the longest intramol. relaxation
time significantly exceeds the shortest exptl. accessible time.
Correspondingly, the shape of the correlation function changes considerably.
Approx. anal. expressions are provided, which are in qual. agreement with the
exact theor. solns. as well as exptl. results, for both DNA and actin
filaments. Our approach is in agreement with the predictions of the Zimm model,
in the limit of very flexible polymers, as well as the predictions of
semiflexible polymer models with respect to the intramol. dynamics in soln.
Woelcke, J., N. Hunt, J. Jungmann
and D. Ullmann. (2006) Early identification of false positives in
high-throughput screening for activators of p53-DNA interaction. Journal of
Biomolecular Screening 11(4):341-350.
Naturally
occurring mutant forms of p53 are deficient for specific DNA binding. However,
their specific DNA binding can be reactivated. The search for small mols. that
reactivate latent p53 is considered to be a cornerstone in cancer therapy. The
authors describe a new homogeneous fluorescent assay approach for the
characterization of binding affinities of human wild-type latent and activated
p53 using DNA*spec(26), with and without the addn. of the antibody PAb421,
resp., and fluorescence correlation spectroscopy (FCS)/2-dimensional
fluorescence-intensity distribution anal. anisotropy as the detection methods.
FCS was compared with 2D-FIDA anisotropy, and a very good correlation of the
results with both readouts was obsd. (KDs for nonspecific DNA binding of
24.4+-3.5 nM with 2D-FIDA anisotropy and of 29.5+-5.5 nM with FCS). The
presence of poly(dI-dC) led to a 10-fold increase of binding affinity (KD of 3.3+-0.5
nM in the presence of PAb421). 2D-FIDA anisotropy was demonstrated to be the
most accurate readout; hence, this detection technol. was selected for a 25,000
compd. member high-throughput screening (HTS) campaign. The hits obtained were
qualified using a novel data evaluation algorithm that identifies false
positives and moreover assesses the validity of true hits in the presence of
the deteriorating artifact. This process step is of utmost importance for
decreasing the attrition in fluorescence-based HTS.
Wu, J. and K. Berland. (2006) Wave
optics analysis of observation volumes in two-photon fluorescence fluctuation
spectroscopy. Proceedings of SPIE-The International Society for Optical
Engineering 6089(Multiphoton Microscopy in the Biomedical Sciences
VI):60890M/1-60890M/7.
Information
recovery in fluorescence fluctuation spectroscopy requires accurate models both
for the phys. dynamics obsd. and for the effective size and shape of the sample
region from which fluorescence signals are measured. In both one- and
two-photon fluctuation spectroscopy, the so called observation vol. is assumed
to be well approximated by a three dimensional Gaussian (3DG) function. Here,
we present wave optics computations that provide an accurate representation of
the true profile for the fluorescence measurement with two-photon excitation.
Fluorescence correlation spectroscopy (FCS) curves are computed for these true
profiles for a variety of optical configurations, and we demonstrate that under
most illumination conditions the 3DG based FCS models do provide reasonable
approxns. to the measured FCS curves.
Xiong, Y., B. Chen, H. S. Smallwood,
R. J. Bieber Urbauer, L. M. Markille, N. Galeva, T. D. Williams and T. C.
Squier. (2006) High-Affinity and Cooperative Binding of Oxidized Calmodulin
by Methionine Sulfoxide Reductase. Biochemistry 45(49):14642-14654.
Methionines
can play an important role in modulating protein-protein interactions assocd.
with intracellular signaling, and their reversible oxidn. to form methionine
sulfoxides [Met(O)] in calmodulin (CaM) and other signaling proteins has been
suggested to couple cellular redox changes to protein functional changes
through the action of methionine sulfoxide reductases (Msr). Prior measurements
indicate the full recovery of target protein activation upon the stereospecific
redn. of oxidized CaM by MsrA, where the formation of the S-stereoisomer of
Met(O) selectively inhibits the CaM-dependent activation of the Ca-ATPase.
However, the physiol. substrates of MsrA remain unclear, as neither the binding
specificities nor affinities of protein targets have been measured. To assess
the specificity of binding and its possible importance in the maintenance of
CaM function, we have measured the kinetics of repair and the binding affinity
between oxidized CaM and MsrA. Redn. of Met(O) in fully oxidized CaM by MsrA is
sensitive to the protein fold, as repair of the intact protein is incomplete,
with >6 Met(O) remaining in each CaM following MsrA redn. In contrast,
following proteolytic digestion, MsrA is able to fully reduce one-half of the
oxidized methionines, indicating that surface-accessible Met(O) within folded
proteins need not be substrates for MsrA repair. Mutation of the active site
(i.e., C72S) in MsrA permitted equil.-binding measurements using both ensemble
and single-mol. fluorescence correlation spectroscopy measurements. We observe
cooperative binding of two MsrA to each CaMox with an apparent affinity (K = 70
+- 10 nM) that is 3 orders of magnitude greater than the Michaelis const. (KM =
68 +- 4 mM). The high-affinity and cooperative interaction between MsrA and
CaMox suggests an important regulatory role of MsrA in the binding and redn. of
Met(O) in functionally sensitive proteins, such that multiple MsrA proteins are
recruited to simultaneously bind and reduce Met(O) in highly oxidized proteins.
Given the suggested role of Met(O) in modulating reversible binding
interactions between proteins assocd. with cellular signaling, these results
indicate an ability of MsrA to selectively reduce Met(O) within highly
surface-accessible sequences to maintain cellular function as part of an
adaptive response to oxidative stress.
Yao, J., K. M. Munson, W. W. Webb
and J. T. Lis. (2006) Dynamics of heat shock factor association with native
gene loci in living cells. Nature (London, United Kingdom) 442(7106):1050-1053.
Direct
observation of transcription factor action in the living cell nucleus can
provide important insights into gene regulatory mechanisms. Live-cell imaging
techniques have enabled the visualization of a variety of intranuclear
activities, from chromosome dynamics to gene expression. However, progress in
studying transcription regulation of specific native genes has been limited,
primarily as a result of difficulties in resolving individual gene loci and in
detecting the small no. of protein mols. functioning within active
transcription units. Here the authors report that multiphoton microscopy
imaging of polytene nuclei in living Drosophila salivary glands allows real-time
anal. of transcription factor recruitment and exchange on specific native
genes. After heat shock, the authors have visualized the recruitment of RNA
polymerase II (Pol II) to native hsp70 gene loci 87A and 87C in real time. The
authors show that heat shock factor (HSF), the transcription activator of
hsp70, is localized to the nucleus before heat shock and translocates from
nucleoplasm to chromosomal loci after heat shock. Assays based on fluorescence
recovery after photobleaching show a rapid exchange of HSF at chromosomal loci
under non-heat-shock conditions but a very slow exchange after heat shock.
However, this is not a consequence of a change of HSF diffusibility, as shown
here directly by fluorescence correlation spectroscopy. These results provide
strong evidence that activated HSF is stably bound to DNA in vivo and that
turnover or disassembly of transcription activator is not required for rounds
of hsp70 transcription. This and previous studies indicate that transcription
activators display diverse dynamic behaviors in their assocns. with targeted
loci in living cells. This method can be applied to study the dynamics of many
factors involved in transcription and RNA processing, and in their regulation
at native heat shock genes in vivo.
Yeh, H.-C., C. M. Puleo, T. C. Lim,
Y.-P. Ho, P. E. Giza, R. C. C. Huang and T.-H. Wang. (2006) A
microfluidic-FCS platform for investigation on the dissociation of Sp1-DNA
complex by doxorubicin. Nucleic Acids Research 34(21):e144/1-e144/9.
The
transcription factor (TF) Sp1 is a well-known RNA polymerase II transcription
activator that binds to GC-rich recognition sites in a no. of essential
cellular and viral promoters. In addn., direct interference of Sp1 binding to
DNA cognate sites using DNA-interacting compds. may provide promising therapies
for suppression of cancer progression and viral replication. In this study, we
present a rapid, sensitive and cost-effective evaluation of a GC intercalative
drug, doxorubicin (DOX), in dissocg. the Sp1-DNA complex using fluorescence
correlation spectroscopy (FCS) in a microfluidic system. FCS allows assay
miniaturization without compromising sensitivity, making it an ideal anal.
method for integration of binding assays into high-throughput, microfluidic
platforms. A polydimethylsiloxane (PDMS)-based microfluidic chip with a mixing
network is used to achieve specific drug concns. for drug titrn. expts. Using
FCS measurements, the IC50 of DOX on the dissocn. of Sp1-DNA complex is estd.
to be 0.55 mM, which is comparable to that measured by the electrophoretic
mobility shift assay (EMSA). However, completion of one drug titrn. expt. on
the proposed microfluidic-FCS platform is accomplished using only picograms of
protein and DNA samples and less than 1 h total assay time, demonstrating vast
improvements over traditional ensemble techniques.
Yu, L., M. Tan, B. Ho, J. L. Ding
and T. Wohland. (2006) Determination of critical micelle concentrations and
aggregation numbers by fluorescence correlation spectroscopy: Aggregation of a
lipopolysaccharide. Analytica Chimica Acta 556(1):216-225.
Fluorescence
correlation spectroscopy (FCS) is often used to det. the mass or radius of a
particle by using the dependence of the diffusion coeff. on the mass and shape.
In this article we discuss how the particle size of aggregates can be measured
by using the concn. dependence of the amplitude of the autocorrelation function
(ACF) instead of the temporal decay. We titrate a soln. of aggregates or
micelles with a fluorescent label that possesses a high affinity for these
structures and measure the changes in the amplitude of the ACF. We develop the
theory describing the change of the ACF amplitude with increasing concns. of
labels and use it to fit exptl. data. It is shown how this method can det. the
aggregation no. and crit. micelle concn. of a std. detergent nonaethylene
glycol monododecyl ether (C12E9) and a lipopolysaccharide (LPS: Escherichia
coli 0111:B4).
Yue, H. and D. H. Waldeck. (2006) Hydrodynamic
and kinetic properties of conjugated polyelectrolytes studied with fluorescence
correlation spectroscopy (FCS). Polymer Preprints (American Chemical
Society, Division of Polymer Chemistry) 47(2):526-527.
The
diffusion of polyethynylbenzenes PEB under various soln. conditions were studied
with FCS. FCS is an effective single mol. method for characterizing the
hydrodynamic properties of fluorescent polymers. The FCS data combined with
steady-state fluorescence spectra indicate that aggregation of conjugated
polyelectrolyte like polyethynylbenzenes can be facilitated by electronic
excitation. Their aggregation is also strongly affected by the solute-solvent
interactions. A direct interaction exists between PEB and surfactant PEG, but
its nature is still unclear.
Ziechner, U., R. Schoenherr, A.-K.
Born, O. Gavrilova-Ruch, R. W. Glaser, M. Malesevic, G. Kuellertz and S. H.
Heinemann. (2006) Inhibition of human ether a go-go potassium channels by
Ca2+/calmodulin binding to the cytosolic N- and C-termini. FEBS Journal 273(5):1074-1086.
Human ether a go-go potassium channels (hEAG1) open in response to membrane depolarization, and they are inhibited by Ca2+/calmodulin (CaM), presumably binding to the C-terminal domain of the channel subunits. Deletion of the cytosolic N-terminal domain resulted in complete abolition of Ca2+/CaM sensitivity suggesting the existence of further CaM binding sites. A peptide array-based screen of the entire cytosolic protein of hEAG1 identified three putative CaM-binding domains, two in the C-terminus (BD-C1: 674-683, BD-C2: 711-721) and one in the N-terminus (BD-N: 151-165). Binding of GST-fusion proteins to Ca2+/CaM was assayed with fluorescence correlation spectroscopy, surface plasmon resonance spectroscopy and pptn. assays. In the presence of Ca2+, BD-N and BD-C2 provided dissocn. consts. in the nanomolar range, BD-C1 bound with lower affinity. Mutations in the binding domains reduced inhibition of the functional channels by Ca2+/CaM. Employment of CaM-EF hand mutants showed that CaM binding to the N- and C-terminus are primarily dependent on EF hand motifs 3 and 4. Hence, closure of EAG channels presumably requires the binding of multiple CaM mols. in a manner more complex than previously assumed.