|
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