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2004 FCS papers |
Interaction of mucin
with cholesterol enriched vesicles: role of mucin structural domains. Afdhal, N.H., Cao, X., Bansil, R.,
Hong, Z., Thompson, C., Brown, B., and Wolf, D., Biomacromolecules,
2004. 5(2): pp. 269-75. We utilized fluorescence recovery after
photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) to
examine the role of gallbladder mucin (GBM) in promoting the aggregation and/or
fusion of cholesterol enriched vesicles. By fluorescent labeling either the
vesicle or the mucin, we could examine the change in vesicle size as well as
changes in mucin's diffusion constant. Both FRAP and FCS show that GBM has a
profound effect in inducing vesicles to aggregate/fuse, particularly after
overnight incubation. GBM mucin domains (either protease digested or reduced
GBM) are not as effective as native GBM. Intact GBM alone was able to shorten
crystal appearance time and increase the number of crystals nucleated by
polarized optical microscopy. In summary, our findings would suggest that both
glycosylated and nonglycosylated domains of GBM are involved in early
aggregation of cholesterol enriched vesicles but that this effect is reversible
in the absence of nonglycosylated domains.
Comparative study of
the haplotype structure and linkage disequilibrium of chromosome 1p36.2 region
in the Korean and Japanese populations. Akesaka, T., Lee, S.G., Ohashi, J., Bannai, M., Tsuchiya, N.,
Yoon, Y., Tokunaga, K., and Song, K., J Hum Genet, 2004. 49(11):
pp. 603-9. The patterns of linkage disequilibrium (LD) in the human genome
provide important information for disease gene mapping. LDs may vary depending
on chromosomal regions and populations. We have compared LD and haplotypes
defined by SNPs in the chromosome 1p36.2 region of the Korean and Japanese
populations. Fifty-eight SNPs in about 418 kb ranging from tumor necrosis
factor receptor 2 (TNFR2:TNFRSF1B) to procollagen-lysine, 2-oxoglutarate
5-dioxygenase (PLOD) gene were examined in 96 healthy Koreans and Japanese each
by direct sequencing and fluorescence correlation spectroscopy combined with
the PCR-sequence specific primer method (PCR-SSP-FCS), respectively. Upon
pair-wise LD analysis, a total of 25 and 16 out of 58 SNPs greater than MAF 10%
were included in LD blocks, encompassing almost 81 kb and 55 kb in total, in
Koreans and Japanese, respectively. Both similarities and differences were
observed in LD strength and haplotype frequencies between the populations.
Considerable similarities were observed in the telomeric region where a
long-range block of approximately 80 kb including three genes was found to have
strong LDs in both Koreans and Japanese. Significant difference in LD strength
was present near the TNFR2 region between the Japanese and Korean populations.
Fluorescence
correlation spectroscopy relates rafts in model and native membranes. Bacia, K., Scherfeld, D., Kahya,
N., and Schwille, P., Biophys J, 2004. 87(2): pp. 1034-43. The
lipid raft model has evoked a new perspective on membrane biology.
Understanding the structure and dynamics of lipid domains could be a key to
many crucial membrane-associated processes in cells. However, one shortcoming
in the field is the lack of routinely applicable techniques to measure raft
association without perturbation by detergents. We show that both in cell and
in domain-exhibiting model membranes, fluorescence correlation spectroscopy
(FCS) can easily distinguish a raft marker (cholera toxin B subunit bound to
ganglioside (GM1) and a nonraft marker (dialkylcarbocyanine dye diI)) by their
decidedly different diffusional mobilities. In contrast, these markers exhibit
only slightly different mobilities in a homogeneous artificial membrane.
Performing cholesterol depletion with methyl-beta-cyclodextrin, which disrupts
raft organization, we find an analogous effect of reduced mobility for the
nonraft marker in domain-exhibiting artificial membranes and in cell membranes.
In contrast, cholesterol depletion has differential effects on the raft marker,
cholera toxin B subunit-GM1, rendering it more mobile in artificial
domain-exhibiting membranes but leaving it immobile in cell membranes, where
cytoskeleton disruption is required to achieve higher mobility. Thus,
fluorescence correlation spectroscopy promises to be a valuable tool to
elucidate lipid raft associations in native cells and to gain deeper insight
into the correspondence between model and natural membranes.
Characterizing
specific phage-protein interactions by fluorescence correlation spectroscopy. Bahns, J.T., Liu, C.M., and Chen,
L., Protein Sci, 2004. 13(10): pp. 2578-87. The interactions of
several affinity reagent displayed T7 and M13 phage particles with their
corresponding target molecules were examined using Fluorescence Correlation
Spectroscopy (FCS). Diffusion times, relative fractions of each component in
the recognition reactions at the equilibrium state, and ultimately the
dissociation constants were deduced from analyzing the fluorescence
autocorrelation curves. Although the sample preparation and FCS
characterization of icosahedral T7-related systems were relatively straight
forward, procedures with filamentous M13-related systems were complicated by
the physical size of M13 and its aggregate formation. Methods that accommodate
the FCS measurement of the M13 phage via changing confocal optics, fitting
procedures, and aggregate discrimination are presented and discussed.
Single-nucleotide-polymorphism
genotyping for whole-genome-amplified samples using automated fluorescence
correlation spectroscopy. Bannai, M., Higuchi, K., Akesaka, T., Furukawa, M., Yamaoka, M., Sato,
K., and Tokunaga, K., Anal Biochem, 2004. 327(2): pp. 215-21.
Whole-genome amplification (WGA) methods were adopted for
single-nucleotide-polymorphism (SNP) typing to minimize the amount of genomic
DNA that has to be used in typing for thousands of different SNPs in
large-scale studies; 5-10 ng of genomic DNA was amplified by a WGA method
(improved primer-extension-preamplification-polymerase chain reaction
(I-PEP-PCR), degenerated oligonucleotide primer-PCR (DOP-PCR), or multiple
displacement amplification (MDA)). Using 1/100 to 1/500 amounts of the
whole-genome-amplified products as templates, subsequent analyses were
successfully performed. SNPs were genotyped by the sequence-specific primer
(SSP)-PCR method followed by fluorescence correlation spectroscopy (FCS). The
typing results were evaluated for four different SNPs on tumor necrosis factor
receptor 1 and 2 genes (TNFR1 and TNFR2). The genotypes determined by the
SSP-FCS method using the WGA products were 100% in concordance with those
determined by nucleotide sequencing using genomic DNAs. We have already carried
out typing of more than 300 different SNPs and are currently performing
7,500-10,000 typings per day using WGA samples from patients with several
common diseases. WGA coupled with FCS allows specific and high-throughput
genotyping of thousands of samples for thousands of different SNPs.
Polarized fluorescence
correlation spectroscopy of DNA-DAPI complexes. Barcellona, M.L., Gammon, S.,
Hazlett, T., Digman, M.A., and Gratton, E., Microsc Res Tech, 2004. 65(4-5):
pp. 205-17. We discuss the use of fluorescence correlation spectroscopy for the
measurement of relatively slow rotations of large macromolecules in solution or
attached to other macromolecular structures. We present simulations and
experimental results to illustrate the range of rotational correlation times
and diffusion times that the technique can analyze. In particular, we examine
various methods to analyze the polarization fluctuation data. We have found
that by first constructing the polarization function and then calculating the
autocorrelation function, we can obtain the rotational motion of the molecule
with very little interference from the lateral diffusion of the macromolecule,
as long as the rotational diffusion is significantly faster than the lateral
diffusion. Surprisingly, for common fluorophores the autocorrelation of the polarization
function is relatively unaffected by the photon statistics. In our instrument,
two-photon excitation is used to define a small volume of illumination where a
few molecules are present at any instant of time. The measurements of long DNA
molecules labeled with the fluorescent probe DAPI show local rotational motions
of the polymers in addition to translation motions of the entire polymer. For
smaller molecules such as EGFP, the viscosity of the solution must be increased
to bring the relaxation due to rotational motion into the measurable range.
Overall, our results show that polarized fluorescence correlation spectroscopy
can be used to detect fast and slow rotational motion in the time scale from
microsecond to second, a range that cannot be easily reached by conventional
fluorescence anisotropy decay methods.
Detection of specific
DNA sequences using dual-color two-photon fluorescence correlation spectroscopy. Berland, K.M., J Biotechnol,
2004. 108(2): pp. 127-36. Fluorescence correlation spectroscopy (FCS) is
rapidly growing in popularity as a biomedical research tool. FCS measurements
can produce an accurate characterization of the chemical, physical, and kinetic
properties of a biological system. They can also serve as a diagnostic,
detecting particular molecular species with high sensitivity and specificity.
We here demonstrate that dual-color FCS measurements can be applied to detect
and quantify the concentration of specific non-fluorescent molecular species
without requiring any modifications to the molecule of interest. We demonstrate
this capability by applying dual-color two-photon fluorescence
cross-correlation spectroscopy to detect single stranded gamma tubulin DNA in
solution with high sensitivity. This quantification is independent of molecular
size, and the methods introduced can be extended to measurements in complex
environments such as within living cells.
Fluorescence
correlation spectroscopy: a new tool for quantification of molecular
interactions.
Berland, K.M., Methods Mol Biol, 2004. 261: pp. 383-98.
Fluorescence correlation spectroscopy (FCS) provides a powerful method to
measure molecular dynamics and interactions in a wide variety of experimental
systems and environments. In this article we focus on the use of FCS methods to
quantify molecular interactions, including the use of diffusion analysis and
molecular counting. Both autocorrelation and cross-correlation FCS measurements
are discussed.
Characterization of
simian virus 40 on its infectious entry pathway in cells using fluorescence
correlation spectroscopy. Bernacchi, S., Mueller, G., Langowski, J., and Waldeck, W., Biochem
Soc Trans, 2004. 32(Pt 5): pp. 746-9. SV40 (simian virus 40) is a
double-stranded DNA virus and is strongly oncogenic in experimental animals.
SV40 enters cells by atypical endocytosis mediated by caveolae, transporting
the virus to its usual destination, namely the endoplasmic reticulum. The
cellular mechanisms of capsid disassembly (uncoating) and deliverance of the
viral genome into the cellular nucleus remain unknown. Here, we study (i) the
formation of caveolae after viral infection and the diffusion of caveosome
vesicles in the cytoplasm and (ii) the capsid disassembly and the mobility of
the viral genome on its way to the nucleus, using fluorescence correlation
spectroscopy. To follow the viral genome and capsids separately, the histone
components of SV40 minichromosomes were labelled with enhanced yellow
fluorescent protein and the capsid was labelled with a fluorescent red dye,
Alexa568. We characterized the diffusion of caveosomes, the capsid disassembly
process in the cytoplasm and the mobility of the viral genome in the nucleus,
using two kinds of permissive cells.
The mobility of
phytochrome within protonemal tip cells of the moss Ceratodon purpureus,
monitored by fluorescence correlation spectroscopy. Bose, G., Schwille, P., and
Lamparter, T., Biophys J, 2004. 87(3): pp. 2013-21. Fluorescence
correlation spectroscopy (FCS) is a versatile tool for investigating the
mobilities of fluorescent molecules in cells. In this article, we show that it
is possible to distinguish between freely diffusing and membrane-bound forms of
biomolecules involved in signal transduction in living cells. Fluorescence
correlation spectroscopy was used to measure the mobility of phytochrome, which
plays a role in phototropism and polarotropism in protonemal tip cells of the
moss Ceratodon purpureus. The phytochrome was loaded with phycoerythrobilin,
which is fluorescent only in the phytochrome-bound state. Confocal laser
scanning microscopy was used for imaging and selecting the xy measuring
position in the apical zone of the tip cell. Fluorescence correlation was
measured at ancient z-positions in the cell. Analysis of the diffusion
coefficients by nonlinear least-square fits showed a subcellular fraction of
phytochrome at the cell periphery with a sixfold higher diffusion coefficient
than in the core fraction. This phytochrome is apparently bound to the membrane
and probably controls the phototropic and polarotropic response.
Hydrodynamics of
nanoscopic tubulin rings in dilute solutions. Boukari, H., Nossal, R., Sackett, D.L., and Schuck,
P., Phys Rev Lett, 2004. 93(9): p. 098106. We combine
fluorescence correlation spectroscopy and sedimentation velocity measurements to
probe the hydrodynamic behavior of tubulin dimers and nanoscopic tubulin rings.
The rings are rigid, have circular geometry, and are monodisperse in size. We
use the high-precision ratio of the sedimentation coefficients and that of the
translational diffusion coefficients to validate theories for calculating the
hydrodynamic properties of supramolecular structures.
Quantitative analysis
of the formation and diffusion of A1-adenosine receptor-antagonist complexes in
single living cells.
Briddon, S.J., Middleton, R.J., Cordeaux, Y., Flavin, F.M., Weinstein, J.A.,
George, M.W., Kellam, B., and Hill, S.J., Proc Natl Acad Sci U S A,
2004. 101(13): pp. 4673-8. The A1-adenosine receptor (A1-AR) is a G
protein-coupled receptor that mediates many of the physiological effects of
adenosine in the brain, heart, kidney, and adipocytes. Currently, ligand
interactions with the A1-AR can be quantified on large cell populations only by
using radioligand binding. To increase the resolution of these measurements, we
have designed and characterized a previously undescribed fluorescent antagonist
for the A1-AR, XAC-BY630, based on xanthine amine congener (XAC). This compound
has been used to quantify ligand-receptor binding at a single cell level using
fluorescence correlation spectroscopy (FCS). XAC-BY630 was a competitive
antagonist of A1-AR-mediated inhibition of cAMP accumulation [log10 of the
affinity constant (pKb) = 6.7)] and stimulation of inositol phosphate
accumulation (pKb = 6.5). Specific binding of XAC-BY630 to cell surface A1-AR
could also be visualized in living Chinese hamster ovary (CHO)-A1 cells by
using confocal microscopy. FCS analysis of XAC-BY630 binding to the membrane of
CHO-A1 cells revealed three components with diffusion times (tauD) of 62 micros
(tauD1, free ligand), 17 ms (tauD2, A1-AR-ligand), and 320 ms (tauD3).
Confirmation that tauD2 resulted from diffusion of ligand-receptor complexes
came from the similar diffusion time observed for the fluorescent A1-AR-Topaz
fusion protein (15 ms). Quantification of tauD2 showed that the number of
receptor-ligand complexes increased with increasing free ligand concentration
and was decreased by the selective A1-AR antagonist,
8-cyclopentyl-1,3-dipropylxanthine. The combination of FCS with XAC-BY630 will
be a powerful tool for the characterization of ligand-A1-AR interactions in
single living cells in health and disease.
Application of
fluorescence correlation spectroscopy to the measurement of agonist binding to
a G-protein coupled receptor at the single cell level. Briddon, S.J., Middleton, R.J.,
Yates, A.S., George, M.W., Kellam, B., and Hill, S.J., Faraday Discuss,
2004. 126: pp. 197-207; discussion 245-54. The A1-adenosine receptor
(A1-AR) is a member of the G-protein coupled receptor superfamily, which has significant
pathophysiological importance in disorders such as heart arrhythmias, asthma
and stroke. Here, we have used fluorescence correlation spectroscopy (FCS) to
facilitate the study of A1-AR pharmacology at the subcellular level. To this
end, we have successfully designed and synthesised a fluorescently labelled
A1-AR agonist, ABA-BY630. ABA-BY630 is an N6- derivative of adenosine
conjugated to the red-excited fluorophore, BODIPY" 630/650. In CHO cells
expressing the human A1-AR, ABA-BY630 shows strong and potent agonist activity
at this receptor. Specific binding of ABA-BY630 to the A1-AR in cell membranes
of living CHO cells can also be visualised using confocal microscopy. Moreover,
using FCS, we can detect and quantify the binding of ABA-BY630 to the A1-AR in
a small area (0.2 microm2) of the upper cell membrane. FCS measurements
indicate the presence of at least two populations of receptor-ABA-BY630
complexes with diffusion times of 8 and 233 ms. The quantity of both of these
complexes was significantly reduced by pre-incubation with the A1-AR antagonist
DPCPX. Application of FCS in conjunction with ABA-BY630 will allow the
comparison of A1-AR pharmacology in single cells from healthy and diseased
tissue.
Measuring unfolding
of proteins in the presence of denaturant using fluorescence correlation
spectroscopy.
Chattopadhyay, K., Saffarian, S., Elson, E.L., and Frieden, C., Biophys J,
2005. 88(2): pp. 1413-22. IFABP is a small (15 kDa) protein consisting
mostly of antiparallel beta-strands that surround a large cavity into which
ligands bind. We have previously used FCS to show that the native protein,
labeled with fluorescein, exhibits dynamic fluctuation with a relaxation time
of 35 micros. Here we report the use of FCS to study the unfolding of the protein
induced by guanidine hydrochloride. Although the application of this technique
to measure diffusion coefficients and molecular dynamics is straightforward,
the FCS results need to be corrected for both viscosity and refractive index
changes as the guanidine hydrochloride concentration increases. We present here
a detailed study of the effects of viscosity and refractive index of guanidine
hydrochloride solutions to calibrate FCS data. After correction, the increase
in the diffusion time of IFABP corresponds well with the unfolding transition
monitored by far ultraviolet circular dichroism. We also show that the
magnitude of the 35 micros phase, reflecting the conformational fluctuation in
the native state, decreases sharply as the concentration of denaturant
increases and the protein unfolds. Although FCS experiments indicate that the
unfolded state at pH 2 is rather compact and native-like, the radius in the
presence of guanidine hydrochloride falls well within the range expected for a
random coil.
Dual-color
photon-counting histogram. Chen, Y., Tekmen, M., Hillesheim, L., Skinner, J., Wu, B., and Muller,
J.D., Biophys J, 2005. 88(3): pp. 2177-92. We report on the
development of dual-color photon-counting histogram (PCH) analysis. Dual-color
PCH is an extension of regular PCH and considers the photon counts received in
two detection channels instead of one. Because each detection channel records a
different color, dual-color PCH distinguishes fluorescent species not only by
differences in their brightness, but also according to their color. The
additional discrimination by color increases the sensitivity of PCH in
resolving a mixture of species considerably. Most dual-color fluorescence
fluctuation experiments are performed on fluorophores with overlapping emission
spectra. This overlap results in spectral cross talk between the detector
channels, which reduces resolvability. Here, we demonstrate that dual-color PCH
is able to resolve binary dye mixtures in the presence of cross talk from a
single measurement without any additional information about the sample. We
discuss the effect of sampling time on the fit parameters of dual-color PCH.
Differences between dual-color fluorescence correlation spectroscopy and
dual-color PCH will also be addressed. We quantitatively resolve a mixture of
the two fluorescent proteins CFP and YFP, which is challenging because of the
strong spectral overlap of their emission spectra. Dichroic mirrors are needed
to direct the light into the two detection channels. We quantify the influence
of these filters on dual-color PCH analysis and determine the optimal
transition wavelength of the dichroic mirror for the CFP-YFP pair.
Mechanism of protein
binding to spherical polyelectrolyte brushes studied in situ using two-photon
excitation fluorescence fluctuation spectroscopy. Czeslik, C., Jansen, R., Ballauff,
M., Wittemann, A., Royer, C.A., Gratton, E., and Hazlett, T., Phys Rev E
Stat Nonlin Soft Matter Phys, 2004. 69(2 Pt 1): p. 021401. We used
two-photon excitation fluorescence fluctuation spectroscopy with photon
counting histogram (PCH) analysis as a new tool to study the binding of
globular proteins to colloidal particles in situ. Whereas fluorescence
fluctuations are traditionally evaluated by calculating the autocorrelation function
(fluorescence correlation spectroscopy), a complementary PCH analysis has been
performed in this study which is advantageous when particle concentrations of a
multicomponent system are of interest and the particles can be distinguished
through particle brightness differences. The binding of two proteins,
staphylococcal nuclease (SNase) and bovine serum albumin (BSA), to spherical
polyelectrolyte brushes (SPB) was measured as a function of protein
concentration and ionic strength of the solution at pH-values where SNase and
BSA are positively and negatively charged, respectively. It has been found that
SNase and BSA strongly bind to the SPB regardless of the protein charge. When
the ionic strength of the solution is raised to 100 mM, the SPB become resistant
to both proteins. These findings provide further evidence for a binding
mechanism where the proteins are mainly driven to the SPB by the
"counterion evaporation" force, while Coulomb interactions play a
minor role. The results of this study characterize the potential of SPB as a
new class of carrier particles for proteins whose use in biotechnological
applications appears to be rewarding.
Molecular crowding
reduces to a similar extent the diffusion of small solutes and macromolecules:
measurement by fluorescence correlation spectroscopy. Dauty, E. and Verkman, A.S., J
Mol Recognit, 2004. 17(5): pp. 441-7. Aqueous environments in living
cells are crowded, with up to >50 wt% small and macromolecule-size solutes.
We investigated quantitatively one important consequence of molecular
crowding--reduced diffusion of biologically important solutes. Fluorescence
correlation spectroscopy (FCS) was used to measure the diffusion of a series of
fluorescent small solutes and macromolecules. In water, diffusion coefficients
(D(o)w) were (in cm2/s x 10(-8)): rhodamine green (270), albumin (52), dextrans
(75, 10 kDa; 10, 500 kDa), double-stranded DNAs (96, 20 bp; 10, 1 kb; 3.4, 4.5
kb) and polystyrene nanospheres (5.4, 20 nm diameter; 2.3, 100 nm).
Aqueous-phase diffusion (Dw) in solutions crowded with Ficoll-70 (0-60 wt%) was
reduced by up to 650-fold in an exponential manner: Dw = D(o)w exp
(-[C]/[C]exp), where [C]exp is the concentration (in wt%) of crowding agent
reducing D(o)w by 63%. FCS data for all solutes and Ficoll-70 concentrations
fitted well to a model of single-component, simple (non-anomalous) diffusion.
Interestingly [C]exp were nearly identical (11+/-2 wt%, SD) for diffusion of
the very different types of macromolecules in Ficoll-70 solutions. However, [C]exp
was dependent on the nature of the crowding agent: for example, [C]exp for
diffusion of rhodamine green was 30 wt% for glycerol and 16 wt% for 500 kDa
dextran. Our results indicate that molecular crowding can greatly reduce
aqueous-phase diffusion of biologically important macromolecules, and
demonstrate a previously unrecognized insensitivity of crowding effects on the
size and characteristics of the diffusing species.
Photobleaching,
mobility, and compartmentalisation: inferences in fluorescence correlation
spectroscopy.
Delon, A., Usson, Y., Derouard, J., Biben, T., and Souchier, C., J Fluoresc,
2004. 14(3): pp. 255-67. In living cells the transport and diffusion of
molecules is constrained by compartments of various sizes. This paper is an
attempt to show that the size of these compartments can in principle be
estimated experimentally from Fluorescence Correlation Spectroscopy (FCS)
combined with the measurement of the photobleaching rate. In this work,
confocal fluorescence microscopy experiments have been carried out on giant
unilamellar vesicles, a system that mimics cellular compartmentalisation. We
have developed numerical and analytical models to describe the fluorescence
decay due to photobleaching in this geometry, which has enabled us to point out
two regimes depending on the value of the parameter P(B) = sigma(B)P/D (where
sigma(B) is the photobleaching cross section of the dye, D its diffusion
constant, and P the laser power (in photon/s)). In particular, when P(B)
<< 1 (i.e. in the fast diffusion regime), the photobleaching rate is
independent of the diffusion constant and scales like sigma(B)P/R2, in
agreement with the experimental results. On the other hand, the standard
diffusion models used to analyse the FCS data do not take into account the
effects of the fluorescence decay on the autocorrelation curve. We show here
how to correct the raw data for these drawbacks.
Binding of the
b-subunit in the ATP synthase from Escherichia coli. Diez, M., Borsch, M., Zimmermann,
B., Turina, P., Dunn, S.D., and Graber, P., Biochemistry, 2004. 43(4):
pp. 1054-64. The rotary mechanism of ATP synthase requires a strong binding
within stator subunits. In this work we studied the binding affinity of the
b-subunit to F(1)-ATPase of Escherichia coli. The dimerization of the truncated
b-subunit without amino acids 1-33, b(34-156)T62C, was investigated by
analytical ultracentrifugation, resulting in a dissociation constant of 1.8
microM. The binding of b-subunit monomeric and dimeric forms to the isolated
F(1) part was investigated by fluorescence correlation spectroscopy and
steady-state fluorescence. The mutants b(34-156)T62C and EF(1)-gammaT106C were
labeled with several fluorophores. Fluorescence correlation spectroscopy was
used to measure translational diffusion times of the labeled b-subunit, labeled
F(1), and a mixture of the labeled b-subunit with unlabeled F(1). Data analysis
revealed a dissociation constant of 0.2 nM of the F(1)b(2) complex, yielding a
Gibbs free energy of binding of DeltaG(o)= -55 kJ mol(-1). In steady-state
fluorescence resonance energy transfer (FRET) measurements it was found that
binding of the b-subunit to EF(1)-gammaT106C-Alexa488 resulted in a
fluorescence decrease of one-third of the initial FRET donor fluorescence
intensity. The decrease of fluorescence was measured as a function of
b-concentration, and data were described by a model including equilibria for
dimerization of the b-subunit and binding of b and b(2) to F(1). For a
quantitative description of fluorescence decrease we used two different models:
the binding of the first and the second b-subunit causes the same fluorescence
decrease (model 1) or only the binding of the first b-subunit causes
fluorescence decrease (model 2). Data evaluation revealed a dissociation
constant for the F(1)b(2) complex of 0.6 nM (model 1) or 14 nM (model 2),
giving DeltaG(o)= -52 kJ mol(-1) and DeltaG(o)= -45 kJ mol(-1), respectively.
The maximal DeltaG observed for ATP synthesis in cells is approximately DeltaG=
55 kJ mol(-1). Therefore, the binding energy of the b-subunit seems to be too
low for models in which the free energy for ATP synthesis is accumulated in the
elastic strain between rotor and stator subunits and then transduced to the
catalytic site in one single step. Models in which energy transduction takes
place in at least two steps are favored.
Distribution, lateral
mobility and function of membrane proteins incorporated into giant unilamellar
vesicles. Doeven,
M.K., Folgering, J.H., Krasnikov, V., Geertsma, E.R., van den Bogaart, G., and
Poolman, B., Biophys J, 2005. 88(2): pp. 1134-42. GUVs have been
widely used for studies on lipid mobility, membrane dynamics and lipid domain
(raft) formation, using single molecule techniques like fluorescence
correlation spectroscopy. Reports on membrane protein dynamics in these types
of model membranes are by far less advanced due to the difficulty of
incorporating proteins into GUVs in a functional state. We have used sucrose to
prevent four distinct membrane protein(s) (complexes) from inactivating during
the dehydration step of the GUV-formation process. The amount of sucrose was
optimized such that the proteins retained 100% biological activity, and many
proteo-GUVs were obtained. Although GUVs could be formed by hydration of lipid
mixtures composed of neutral and anionic lipids, an alternate current electric
field was required for GUV formation from neutral lipids. Distribution, lateral
mobility, and function of an ATP-binding cassette transport system, an
ion-linked transporter, and a mechanosensitive channel in GUVs were determined
by confocal imaging, fluorescence correlation spectroscopy, patch-clamp
measurements, and biochemical techniques. In addition, we show that sucrose
slows down the lateral mobility of fluorescent lipid analogs, possibly due to
hydrogen-bonding with the lipid headgroups, leading to larger complexes with
reduced mobility.
Development of
miniaturized competitive immunoassays on a protein chip as a screening tool for
drugs. Du, H., Wu,
M., Yang, W., Yuan, G., Sun, Y., Lu, Y., Zhao, S., Du, Q., Wang, J., Yang, S.,
Pan, M., Wang, S., and Cheng, J., Clin Chem, 2005. 51(2): pp.
368-75. BACKGROUND: Doping in sports has become a serious problem. Gas
chromatography-mass spectrometry (GC-MS) serves as an effective reference
method, but it is limited by low throughput and is therefore not suitable for
large-scale screening. Use of protein chips for high-throughput screening of
all athletes for prohibited substances could become an important complementary
tool to GC-MS. METHODS: We developed a protein chip based on an
aldehyde-activated glass slide containing 10 physically isolated arrays. The
chip was used to screen urine from 1347 athletes for prohibited substances and
to screen a negative control group consisting of 200 females and 120 males.
Urine samples from 66 individuals known to be abusers, provided by the China
Doping Control Center (CDCC), and 129 standard prohibited substances were
tested as positive controls. RESULTS: All 1347 urine samples screened by means
of the protein chips were also subjected to reference analysis by GC-MS at the
CDCC. There was no qualitative difference between the results obtained with the
two methods. The correlation coefficient (r(2)) for the quantitative results
obtained with the protein chip and GC-MS was 0.991. CONCLUSIONS: The protein
chip could be used to screen for a series of 16 prohibited drugs in urine
samples. This system has the potential to become an effective screening method
to test substances prohibited by the International Olympic Committee.
Fluorescence-based
assessment of LRP activity: a comparative study. Durdux, M., Grunwald, D., Gautier,
T., Ronot, X., and Boutonnat, J., Anticancer Res, 2004. 24(2B):
pp. 725-32. BACKGROUND: Broad resistance to anticancer drugs is a major cause of
failure in cancer treatment. The Lung Resistance-related Protein (LRP) is a
protein associated with drug resistance, which is involved in
nucleo-cytoplasmic transport and is known to predict a poor response to
chemotherapy in acute myeloid leukaemia. The only method allowing the detection
of LRP activity is based on radio-labelled daunorubicin incorporation. Our goal
was to develop a fluorescence-based assay to analyse LRP function. MATERIALS
AND METHODS: We used human colon carcinoma cell lines treated with sodium
butyrate (NaB) in order to induce LRP expression. Daunorubicin efflux in
isolated nuclei was measured by flow cytometry, the localization and
quantification of Daunorubicin analysed by confocal laser scanning microscopy
(CLSM) and the diffusion coefficient of this drug estimated by Fluorescence
Correlation Spectrometry (FCS). RESULTS: According to the method using [14C]
Doxorubicin cells incubated with NaB displayed an efflux of Daunorubicin out of
isolated nuclei demonstrated by flow cytometry or CLSM. The FCS method was able
to evaluate kinetics of Daunorubicin molecules in nucleus and cytoplasm and
showed a higher dispersion of Daunorubicin kinetics with cells previously
NaB-treated. This argument is in favour of an increase of nucleo-cytoplasmic
exchange. CONCLUSION: Using CLSM we showed that LRP was able to modify
anticancer drug repartition in the cells. LRP activity assessment needs either
isolated nuclei if flow cytometry is employed, or FCS, and only a few cells may
be analysed.
HIV-1 nucleocapsid
protein binds to the viral DNA initiation sequences and chaperones their
kissing interactions.
Egele, C., Schaub, E., Ramalanjaona, N., Piemont, E., Ficheux, D., Roques, B.,
Darlix, J.L., and Mely, Y., J Mol Biol, 2004. 342(2): pp. 453-66.
The chaperone properties of the human immunodeficiency virus type 1 (HIV-1)
nucleocapsid protein (NC) are required for the two obligatory strand transfer
reactions occurring during viral DNA synthesis. The second strand transfer
relies on the destabilization and the subsequent annealing of the primer
binding site sequences (PBS) at the 3' end of the (-) and (+) DNA strands. To
characterize the binding and chaperone properties of NC on the (-)PBS and
(+)PBS sequences, we monitored by steady-state and time-resolved fluorescence
spectroscopy as well as by fluorescence correlation spectroscopy the
interaction of NC with wild type and mutant oligonucleotides corresponding to
the (-)PBS and (+)PBS hairpins. NC was found to bind with high affinity to the
loop, the stem and the single-stranded protruding sequence of both PBS
sequences. NC induces only a limited destabilization of the secondary structure
of both sequences, activating the transient melting of the stem only during its
"breathing" period. This probably results from the high stability of
the PBS due to the four G-C pairs in the stem. In contrast, NC directs the
formation of "kissing" homodimers efficiently for both (-)PBS and
(+)PBS sequences. Salt-induced dimerization and mutations in the (-)PBS sequence
suggest that these homodimers may be stabilized by two intermolecular G-C
Watson-Crick base-pairs between the partly self-complementary loops. The
propensity of NC to promote the dimerization of partly complementary sequences
may favor secondary contacts between viral sequences and thus, recombination
and viral diversity.
Quick tour of
fluorescence correlation spectroscopy from its inception. Elson, E.L., J Biomed Opt,
2004. 9(5): pp. 857-64. Fluorescence correlation spectroscopy (FCS) was originally
developed in the early 1970s as a way to measure the kinetics of chemical
reactions under zero perturbation conditions. At its inception, the measurement
was difficult due to experimental limitations and was primarily used during the
1970s and 1980s to characterize diffusion. More recently, as a result of
technological advances, FCS measurements have become easier and more versatile.
In addition to measurements of diffusion both in solution and in cells, FCS is
now also used to measure not only chemical reaction kinetics but also extents
of molecular aggregation, the dynamics of photophysical processes,
conformational fluctuations, molecular interactions in solution and in cells,
and has even found application as a pharmaceutical screening method. From its
inception to the present, the contributions of Webb and his coworkers have had
a central and defining role in the development and applications of FCS.
Art and artefacts of
fluorescence correlation spectroscopy. Enderlein, J., Gregor, I., Patra, D., and Fitter, J., Curr
Pharm Biotechnol, 2004. 5(2): pp. 155-61. Fluorescence correlation
spectroscopy (FCS) is an important technique for studying low concentrations of
analyte molecules in solution. The core molecular characteristic that can be
addressed by FCS is the translational diffusion coefficient of the analyte
molecules, which can be used for i.e. studying molecular binding and reactions,
or conformational changes of macromolecules. The present paper discusses
several possible optical and photophysical effects that can influence the
outcome of a FCS measurement and thus can bias the value of the derived
diffusion coefficient.
Toward oxygen binding
curves of single respiratory proteins. Erker, W., Lippitz, M., Basche, T., and Decker, H., Micron,
2004. 35(1-2): pp. 111-3. Oxygen binding curves of single molecules
promise to discriminate between different models describing cooperativity
because load distributions are accessible. Individual tarantula hemocyanins
could be detected by fluorescence correlation spectroscopy using intrinsic
tryptophan fluorescence as sensor of bound oxygen. However, imaging of
immobilized proteins was not possible due to fast photo-bleaching. It is shown
that tetra-methyl-carboxy-rhodamine (TAMRA), commonly used as a fluorescence
label in single-molecule spectroscopy, can also be applied to monitor bound
oxygen. The dye's fluorescence is quenched due to Forster energy transfer to
the oxygenated active sites of hemocyanin.
Size effects on
diffusion processes within agarose gels. Fatin-Rouge, N., Starchev, K., and Buffle, J., Biophys
J, 2004. 86(5): pp. 2710-9. To investigate diffusion processes in
agarose gel, nanoparticles with sizes in the range between 1 and 140 nm have
been tested by means of fluorescence correlation spectroscopy. Understanding
the diffusion properties in agarose gels is interesting, because such gels are
good models for microbial biofilms and cells cytoplasm. The fluorescence
correlation spectroscopy technique is very useful for such investigations due
to its high sensitivity and selectivity, its excellent spatial resolution
compared to the pore size of the gel, and its ability to probe a wide range of
sizes of diffusing nanoparticles. The largest hydrodynamic radius (R(c)) of
trapped particles that displayed local mobility was estimated to be 70 nm for a
1.5% agarose gel. The results showed that diffusion of particles in agarose gel
is anomalous, with a diverging fractal dimension of diffusion when the large
particles become entrapped in the pores of the gel. The latter situation occurs
when the reduced size (R(A)/R(c)) of the diffusing particle, A, is >0.4.
Variations of the fractal exponent of diffusion (d(w)) with the reduced
particle size were in agreement with three-dimensional Monte Carlo simulations
in porous media. Nonetheless, a systematic offset of d(w) was observed in real
systems and was attributed to weak nonelastic interactions between the
diffusing particles and polymer fibers, which was not considered in the Monte
Carlo simulations.
Focal volume
confinement by submicrometer-sized fluidic channels. Foquet, M., Korlach, J., Zipfel,
W.R., Webb, W.W., and Craighead, H.G., Anal Chem, 2004. 76(6):
pp. 1618-26. Microfluidic channels with two lateral dimensions smaller than 1
microm were fabricated in fused silica for high-sensitivity single-molecule
detection and fluorescence correlation spectroscopy. The effective observation
volumes created by these channels are approximately 100 times smaller than
observation volumes using conventional confocal optics and thus enable
single-fluorophore detection at higher concentrations. Increased
signal-to-noise ratios are also attained because the molecules are restricted
to diffuse through the central regions of the excitation volume. Depending on
the channel geometries, the effective dimensionality of diffusion is reduced,
which is taken into account by simple solutions to diffusion models with
boundaries. Driven by electrokinetic forces, analytes could be flowed rapidly
through the observation volume, drastically increasing the rate of detection
events and reducing data acquisition times. The statistical accuracy of
single-molecule characterization is improved because all molecules are counted
and contribute to the analysis. Velocities as high as 0.1 m/s were reached,
corresponding to average molecular residence times in the observation volume as
short as 10 micros. Applications of these nanofabricated devices for
high-throughput, single-molecule detection in drug screening and genomic
analysis are discussed.
Assembly of
fluorescent chimeric virus-like particles of canine parvovirus in insect cells. Gilbert, L., Toivola, J.,
Lehtomaki, E., Donaldson, L., Kapyla, P., Vuento, M., and Oker-Blom, C., Biochem
Biophys Res Commun, 2004. 313(4): pp. 878-87. Canine parvovirus
(CPV) is a small non-enveloped ssDNA virus composed of the viral proteins VP1,
VP2, and VP3 with a T=1 icosahedral symmetry. VP2 is nested in VP1 and the two
proteins are produced by differential splicing of a primary transcript of the
right ORF of the viral genome. The VP2 protein can be further proteolytically
cleaved to form VP3. Previous studies have shown that VP1 and VP3 are
unnecessary for capsid formation and consequently, that VP2 alone is sufficient
for assembly. We have hypothesized that insertion of the enhanced green
fluorescent protein (EGFP) at the N-terminus of VP2 could be carried out
without altering assembly. To investigate the possibility to develop
fluorescent virus-like particles (fVLPs) from such chimeric VP2 proteins, the
corresponding fusion construct was abundantly expressed in insect cells.
Confocal imaging indicated that the EGFP-VP2 fusion product was assembled to
fluorescent capsid-like complexes. In addition, electron micrographs of
purified EGFP-VP2 complexes showed that they displayed a very similar size and
appearance when compared to VP2 VLPs. Further, immunolabelling of purified
EGFP-VP2 VLPs showed the presence of EGFP within the structure. Fluorescence
correlation spectroscopy (FCS) studies confirmed that fVLPs were very similar
in size when compared to authentic CPV. Finally, feeding of mammalian cells
susceptible to CPV infection with these fVLPs indicated that entry and
intracellular trafficking could be observed. In summary, we have developed
fluorescent virus-like nanoparticles carrying a heterologous entity that can be
utilized as a visualization tool to elucidate events related to a canine
parvovirus infection.
Characterization of
interaction between cationic lipid-oligonucleotide complexes and cellular membrane
lipids using confocal imaging and fluorescence correlation spectroscopy. Gordon, S.P., Berezhna, S.,
Scherfeld, D., Kahya, N., and Schwille, P., Biophys J, 2005. 88(1):
pp. 305-16. Complexes formed by cationic liposomes and single-strand oligodeoxynucleotides
(CL-ODN) are promising delivery systems for antisense therapy. ODN release from
the complexes is an essential step for inhibiting activity of antisense drugs.
We applied fluorescence correlation spectroscopy and confocal laser scanning
microscopy to monitor CL-ODN complex interaction with membrane lipids leading
to ODN release. To model cellular membranes we used giant unilamellar vesicles
and investigated the transport of Cy-5-labeled ODNs across DiO-labeled
membranes. For the first time, we directly observed that ODN molecules are
transferred across the lipid bilayers and are kept inside the giant unilamellar
vesicles after release from the carriers. ODN dissociation from the carrier was
assessed by comparing diffusion constants of CL-ODN complexes and ODNs before
complexation and after release. Freely diffusing Cy-5-labeled ODN (16-nt) has
diffusion constant D(ODN) = 1.3 +/- 0.1 x 10(-6) cm2/s. Fluorescence
correlation spectroscopy curves for CL-ODN complexes were fitted with two
components, which both have significantly slower diffusion in the range of
D(CL-ODN) = approximately 1.5 x 10(-8) cm2/s. Released ODN has the mean
diffusion constant D = 1.1 +/- 0.2 x 10(-6) cm2/s, which signifies that ODN is
dissociated from cationic lipids. In contrast to earlier studies, we report
that phosphatidylethanolamine can trigger ODN release from the carrier in the
full absence of anionic phosphatidylserine in the target membrane and that
phosphatidylethanolamine-mediated release is as extensive as in the case of
phosphatidylserine. The presented methodology provides an effective tool for
probing a delivery potential of newly created lipid formulations of CL-ODN
complexes for optimal design of carriers.
Parallel single
molecule detection with a fully integrated single-photon 2x2 CMOS detector
array. Gosch, M.,
Serov, A., Anhut, T., Lasser, T., Rochas, A., Besse, P.A., Popovic, R.S., Blom,
H., and Rigler, R., J Biomed Opt, 2004. 9(5): pp. 913-21. We
present parallel single molecule detection (SMD) and fluorescence correlation
spectroscopy (FCS) experiments with a fully integrated complementary metal
oxide semiconductor (CMOS) single-photon 2x2 detector array. Multifocal
excitation is achieved with a diffractive optical element (DOE). Special
emphasis is placed on parallelization of the total system. The performance of
the novel single-photon CMOS detector is investigated and compared to a
state-of-the-art single-photon detecting module [having an actively quenched
avalanche photodiode (APD)] by measurements on free diffusing molecules at
different concentrations. Despite the order of magnitude lower detection
efficiency of the CMOS detector compared to the state-of-the-art single-photon
detecting module, we achieve single molecule sensitivity and reliably determine
molecule concentrations. In addition, the CMOS detector performance for the
determination of the fraction of slowly diffusing molecules in a primer
solution (two-component analysis) is demonstrated. The potential of this new
technique for high-throughput confocal-detection-based systems is discussed.
The role of
subtype-specific ligand binding and the C-tail domain in dimer formation of
human somatostatin receptors. Grant, M., Patel, R.C., and Kumar, U., J Biol Chem,
2004. 279(37): pp. 38636-43. G-protein-coupled receptors (GPCRs)
represent the largest and most diverse family of cell surface receptors.
Several GPCRs have been documented to dimerize with resulting changes in
pharmacology. We have previously reported by means of photobleaching
fluorescence resonance energy transfer (pbFRET) microscopy and fluorescence
correlation spectroscopic (FCS) analysis in live cells, that human somatostatin
receptor (hSSTR) 5 could both homodimerize and heterodimerize with hSSTR1 in
the presence of the agonist SST-14. In contrast, hSSTR1 remained monomeric when
expressed alone regardless of agonist exposure in live cells. In an effort to
elucidate the role of ligand and receptor subtypes in heterodimerization, we
have employed both pb-FRET microscopy and Western blot on cells stably
co-expressing hSSTR1 and hSSTR5 treated with subtype-specific agonists. Here we
provide evidence that activation of hSSTR5 but not hSSTR1 is necessary for
heterodimeric assembly. This property was also reflected in signaling as shown
by increases in adenylyl cyclase coupling efficiencies. Furthermore, receptor
C-tail chimeras allowed for the identification of the C-tail as a determinant
for dimerization. Finally, we demonstrate that heterodimerization is
subtype-selective involving ligand-induced conformational changes in hSSTR5 but
not hSSTR1 and could be attributed to molecular events occurring at the C-tail.
Understanding the mechanisms by which GPCRs dimerize holds promise for
improvements in drug design and efficacy.
Agonist-dependent
dissociation of human somatostatin receptor 2 dimers: a role in receptor
trafficking. Grant,
M., Collier, B., and Kumar, U., J Biol Chem, 2004. 279(35): pp.
36179-83. G-protein-coupled receptors (GPCRs) represent the largest and most
diverse family of cell surface receptors. Several GPCRs have been documented to
dimerize with resulting changes in pharmacology and signaling. We have
previously reported, by means of photobleaching fluorescence resonance energy
transfer (pbFRET) microscopy and fluorescence correlation spectroscopic
analysis in live cells, that human somatostatin receptor (hSSTR) 5 could both
homodimerize and heterodimerize with hSSTR1 in the presence of the agonist
SST-14. By contrast, hSSTR1 remained monomeric when expressed alone regardless
of agonist exposure in live cells. However, the effect of the agonist on other
hSSTR members remains unknown. Using pbFRET microscopy and Western blot, we
provide evidence for agonist-dependent dissociation of self-associated hSSTR2
stably expressed in CHO-K1 and HEK-293 cells. Furthermore, the dissociation of
the hSSTR2 dimer occurred in a concentration-dependent manner. Moreover,
blocking receptor dissociation using a cross-linker agent perturbed receptor
trafficking. Taking these data together, we suggest that the process of GPCR
dimerization may operate differently, even among members of the same family,
and that receptor dissociation as well as dimerization may be important steps
for receptor dynamics.
Flow profiling of a
surface-acoustic-wave nanopump. Guttenberg, Z., Rathgeber, A., Keller, S., Radler, J.O.,
Wixforth, A., Kostur, M., Schindler, M., and Talkner, P., Phys Rev E Stat
Nonlin Soft Matter Phys, 2004. 70(5 Pt 2): p. 056311. The flow
profile in a capillary gap and the pumping efficiency of an acoustic micropump
employing surface acoustic waves is investigated both experimentally and
theoretically. Ultrasonic surface waves on a piezoelectric substrate strongly
couple to a thin liquid layer and generate a quadrupolar streaming pattern
within the fluid. We use fluorescence correlation spectroscopy and fluorescence
microscopy as complementary tools to investigate the resulting flow profile.
The velocity was found to depend on the applied power approximately linearly
and to decrease with the inverse third power of the distance from the
ultrasound generator on the chip. The found properties reveal acoustic
streaming as a promising tool for the controlled agitation during microarray
hybridization.
Diffusion in
two-component lipid membranes--a fluorescence correlation spectroscopy and
monte carlo simulation study. Hac, A.E., Seeger, H.M., Fidorra, M., and Heimburg, T., Biophys
J, 2005. 88(1): pp. 317-33. Using fluorescence correlation
spectroscopy, calorimetry, and Monte Carlo simulations, we studied diffusion processes
in two-component membranes close to the chain melting transition. The aim is to
describe complex diffusion behavior in lipid systems in which gel and fluid
domains coexist. Diffusion processes in gel membranes are significantly slower
than in fluid membranes. Diffusion processes in mixed phase regions are
therefore expected to be complex. Due to statistical fluctuations the gel-fluid
domain patterns are not uniform in space and time. No models for such diffusion
processes are available. In this article, which is both experimental and
theoretical, we investigated the diffusion in DMPC-DSPC lipid mixtures as a
function of temperature and composition. We then modeled the fluorescence
correlation spectroscopy experiment using Monte Carlo simulations to analyze
the diffusion process. It is shown that the simulations yield a very good
description of the experimental diffusion processes, and that predicted
autocorrelation profiles are superimposable with the experimental curves. We
believe that this study adds to the discussion on the physical nature of rafts
found in biomembranes.
High count rates with
total internal reflection fluorescence correlation spectroscopy. Hassler, K., Anhut, T., Rigler,
R., Gosch, M., and Lasser, T., Biophys J, 2005. 88(1): pp. L01-3.
Single-molecule
spectroscopic methods.
Haustein, E. and Schwille, P., Curr Opin Struct Biol, 2004. 14(5):
pp. 531-40. Being praised for the mere fact of enabling the detection of
individual fluorophores a dozen years ago, single-molecule techniques nowadays
represent standard methods for the elucidation of the structural rearrangements
of biologically relevant macromolecules. Single-molecule-sensitive techniques,
such as fluorescence correlation spectroscopy, allow real-time access to a
multitude of molecular parameters (e.g. diffusion coefficients, concentration
and molecular interactions). As a result of various recent advances, this
technique shows promise even for intracellular applications. Fluorescence
imaging can reveal the spatial localization of fluorophores on nanometer length
scales, whereas fluorescence resonance energy transfer supports a wide range of
different applications, including real-time monitoring of conformational
rearrangements (as in protein folding). Still in their infancy, single-molecule
spectroscopic methods thus provide unprecedented insights into basic molecular
mechanisms.
Dye-labeled
benzodiazepines: development of small ligands for receptor binding studies
using fluorescence correlation spectroscopy. Hegener, O., Jordan, R., and Haberlein, H., J
Med Chem, 2004. 47(14): pp. 3600-5. To investigate benzodiazepine
receptor binding studies by fluorescence correlation spectroscopy (FCS), the
four fluorophores fluorescein, tetramethylrhodamine, Oregon Green 488, and Alexa
532 were coupled to the benzodiazepine Ro 07-1986/602 (Ro). Binding assays to
polyclonal antibodies to benzodiazepines and at the native benzodiazepine
receptor on the membrane of rat hippocampal neurons were established to examine
the dye-labeled ligands for their benzodiazepine character and their binding
behavior. Both the fluorescein and the Oregon Green488 moiety led to a loss of
the benzodiazepine receptor binding of the corresponding Ro derivatives.
Antibody recognition and interactions to the receptor were observed for the
tetramethylrhodamine derivative (K(D) = 96.0 +/- 9.5 nM) but with a high amount
of nonspecific binding at the cell membrane of about 50%. In saturation
experiments a K(D) value of 97.2 +/- 8.5 nM was found for the Alexa Fluor 532
derivative-antibody interaction. Investigation of the binding of this ligand to
the benzodiazepine receptor in FCS cell measurements led to confirmation of
high specific binding behavior with a K(D) value of 9.9 +/- 1.9 nM. A
nonspecific binding of <10% was observed after coincubation with 1 microM of
midazolam. The different properties of the labeled benzodiazepine derivatives
and the requirements of the fluorophore in small dye-labeled ligands in FCS
binding studies, at the membrane of living cells, are discussed.
Dynamics of
beta2-adrenergic receptor-ligand complexes on living cells. Hegener, O., Prenner, L., Runkel,
F., Baader, S.L., Kappler, J., and Haberlein, H., Biochemistry, 2004. 43(20):
pp. 6190-9. The agonist-induced dynamic regulation of the beta(2)-adrenergic
receptor (beta(2)-AR) on living cells was examined by means of fluorescence
correlation spectroscopy (FCS) using a fluorescence-labeled arterenol
derivative (Alexa-NA) in hippocampal neurons and in alveolar epithelial type II
cell line A549. Alexa-NA specifically bound to the beta(2)-AR of neurons with a
K(D) value of 1.29 +/- 0.31 nM and of A549 cells with a K(D) of 5.98 +/- 1.62
nM. The receptor density equaled 4.5 +/- 0.9 microm(-2) in neurons (rho(N)) and
19.9 +/- 2.0 microm(-2) in A549 cells (rho(A549)). Kinetic experiments revealed
comparable on-rate constants in both cell types (k(on) = 0.49 +/- 0.03 s(-1)
nM(-1) in neurons and k(on) = 0.12 +/- 0.02 s(-1) nM(-1) in A549 cells). In
addition to the free ligand diffusing with a D(free) of (2.11 +/- 0.04) x
10(-6) cm(2)/s, in both cell types receptor-ligand complexes with two distinct
diffusion coefficients, D(bound1) (fast lateral mobility) and D(bound2)
(hindered mobility), were observed [D(bound1) = (5.23 +/- 0.64) x 10(-8)
cm(2)/s and D(bound2) = (6.05 +/- 0.23) x 10(-10) cm(2)/s for neurons, and
D(bound1) = (2.88 +/- 1.72) x 10(-8) cm(2)/s and D(bound2) = (1.01 +/- 0.46) x
10(-9) cm(2)/s for A549 cells]. Fast lateral mobility of the receptor-ligand
complex was detected immediately after addition of the ligand, whereas hindered
mobility (D(bound2)) was observed after a delay of 5 min in neurons (up to 38%
of total binding) and of 15-20 min in A549 cells (up to 40% of total binding).
Thus, the receptor-ligand complexes with low mobility were formed during
receptor regulation. Consistently, stimulation of receptor internalization
using the adenylate cyclase activator forskolin shifted the ratio of
receptor-ligand complexes toward D(bound2). Intracellular FCS measurements and
immunocytochemical studies confirmed the appearance of endocytosed
receptor-ligand complexes in the cytoplasm subjacent to the plasma membrane
after stimulation with the agonist terbutaline (1 microM). This regulatory
receptor internalization was blocked after preincubation with propranolol and
with a cholesterol-complexing saponin alpha-hederin.
Partitioning of NaPi
cotransporter in cholesterol-, sphingomyelin-, and glycosphingolipid-enriched
membrane domains modulates NaPi protein diffusion, clustering, and activity. Inoue, M., Digman, M.A., Cheng,
M., Breusegem, S.Y., Halaihel, N., Sorribas, V., Mantulin, W.W., Gratton, E.,
Barry, N.P., and Levi, M., J Biol Chem, 2004. 279(47): pp.
49160-71. In dietary potassium deficiency there is a decrease in the transport
activity of the type IIa sodium/phosphate cotransporter protein (NaPi) despite
an increase in its apical membrane abundance. This novel posttranslational
regulation of NaPi activity is mediated by the increased glycosphingolipid
content of the potassium-deficient apical membrane. However, the mechanisms by
which these lipids modulate NaPi activity have not been determined. We
determined if in potassium deficiency NaPi is increasingly partitioned in
cholesterol-, sphingomyelin-, and glycosphingolipid-enriched microdomains of
the apical membrane and if the increased presence of NaPi in these microdomains
modulates its activity. By using a detergent-free density gradient flotation
technique, we found that 80% of the apical membrane NaPi partitions into the
low density cholesterol-, sphingomyelin-, and GM1-enriched fractions
characterized as "lipid raft" fractions. In potassium deficiency, a
higher proportion of NaPi was localized in the lipid raft fractions. By
combining fluorescence correlation spectroscopy and photon counting histogram
methods for control and potassium-deficient apical membranes reconstituted into
giant unilamellar vesicles, we showed a 2-fold decrease in lateral diffusion of
NaPi protein and a greater than 2-fold increase in size of protein aggregates/clusters
in potassium deficiency. Our results indicate that NaPi protein is localized in
membrane microdomains, that in potassium deficiency a larger proportion of NaPi
protein is present in these microdomains, and that NaPi lateral diffusion is
slowed down and NaPi aggregation/clustering is increased in potassium
deficiency, both of which could be associated with the decreased Na/Pi
cotransport activity in potassium deficiency.
Lipid domain
formation and dynamics in giant unilamellar vesicles explored by fluorescence
correlation spectroscopy. Kahya, N., Scherfeld, D., Bacia, K., and Schwille, P., J Struct
Biol, 2004. 147(1): pp. 77-89. Lipids in eukaryotic cell membranes
have been shown to cluster in "rafts" with different lipid/protein
compositions and molecular packing. Model membranes such as giant unilamellar
vesicles (GUVs) provide a key system to elucidate the physical mechanisms of
raft assembly. Despite the large amount of work devoted to the detection and
characterization of rafts, one of the most important pieces of information
still missing in the picture of the cell membrane is dynamics: how lipids
organize and move in rafts and how they modulate membrane fluidity. This
missing element is of crucial importance for the trafficking at and from the
periphery of the cell regulated by endo- and exocytosis and, in general, for
the constant turnover which redistributes membrane components. Here, we review
studies of combined confocal fluorescence microscopy and fluorescence
correlation spectroscopy on lipid dynamics and organization in rafts assembled
in GUVs prepared from various lipid mixtures which are relevant to the problem
of raft formation.
Intracellular
calmodulin availability accessed with two-photon cross-correlation. Kim, S.A., Heinze, K.G., Waxham,
M.N., and Schwille, P., Proc Natl Acad Sci U S A, 2004. 101(1):
pp. 105-10. The availability and interactions of signaling proteins are tightly
regulated in time and space to produce specific and localized effects. For
calmodulin (CaM), a key transducer of intracellular Ca(2+) signaling, binding
to its variety of targets initiates signaling cascades and regulates its
subcellular localization, thereby making it unavailable for subsequent binding
interactions. Among CaM's numerous targets, Ca(2+)/CaM-dependent protein kinase
II is one of the most striking due to its unique ability to increase its
affinity for CaM by autophosphorylation and to translocate when bound to
Ca(2+)/CaM. Two-photon fluorescence correlation spectroscopy and
cross-correlation spectroscopy were utilized to compare mobility and molecular
interactions between CaM and Ca(2+)/CaM-dependent protein kinase II in solution
and in living cells. These techniques revealed that CaM availability in cells
could be altered by a change in intracellular conditions. Two-photon
fluorescence cross-correlation spectroscopy exemplifies a generally applicable
approach for studying protein-protein interactions in living cells that allows
access to the behavior of signaling molecules within their native environment
to probe for heterogeneities in signaling pathways in different cellular
compartments.
Detection of
protein-DNA interactions in crude cellular extracts by fluorescence correlation
spectroscopy.
Kobayashi, T., Okamoto, N., Sawasaki, T., and Endo, Y., Anal Biochem,
2004. 332(1): pp. 58-66. Fluorescence correlation spectroscopy (FCS) is
a methodology to examine directly the translational diffusion of individual
fluorescence-labeled molecules in solutions. Recent studies using FCS have
quantified various bimolecular reactions without any need for amplification. To
evaluate further the applicability of FCS, we studied the specific binding
between proteins and DNA in crude biological samples. Using an automated FCS
system that was recently developed in our laboratories and is capable of
distinguishing two or more molecular species in a multicomponent analysis, we
detected the binding of two representative transcription factors, activator
protein-1 (AP-1) and nuclear factor kappa B (NF-kappaB), in nuclear extracts of
HeLa cells quantitatively with each sequence-specific DNA. The binding rates of
these specific interactions were markedly augmented when cells were treated
with tumor necrosis factor alpha which is known to activate both AP-1 and
NF-kappaB. We also observed the pyrrolidine-dithiocarbamate-induced reciprocal
regulation of these transcription factors. These results indicated that FCS is
a useful tool for the analysis of complex interactions of transcription factors
with DNA even in crude cellular extracts, suggesting that it is a powerful
methodology for the study of a wide variety of molecular events under various
experimental conditions.
[A study of
interaction of 7-aminoactinomycin D with DNA by fluorescence correlation
spectroscopy].
Kovalev, A.E., Iakovenko, A.A., and Vekshin, N.L., Biofizika, 2004. 49(6):
pp. 1030-7. The investigation of interaction of 7-aminoactinomycin D with DNA
is hampered at nanomolar concentrations by low quantum yield of the dye
fluorescence and high adsorbability of the dye. It was found that, instead of
the increase in the fluorescence of 7-aminoactinomycin D after binding with
DNA, a decrease in the fluorescence takes place, because 7-aminoactinomycin D
complexed with DNA exists in two states: photochemically active and inactive.
The presence of the photochemically active state of 7-aminoactinomycin D causes
an apparent increase in the diffusion coefficient upon embedding of
7-aminoactinomycin D into DNA. The data obtained allow one to state that
7-aminoactinomycin D: (1) forms dimers at micromolar concentration, (2) has two
specific photodestruction times, and (3) binds with DNA more effectively than
actinomycin D. Polarization measurements made it possible to estimate
numerically the adsorption of 7-aminoactinomycin D on the walls of the
measuring cell.
Correct diffusion
coefficients of proteins in fluorescence correlation spectroscopy. Application
to tubulin oligomers induced by Mg2+ and Paclitaxel. Krouglova, T., Vercammen, J., and
Engelborghs, Y., Biophys J, 2004. 87(4): pp. 2635-46. In view of
recent warnings for artifacts in fluorescence correlation spectroscopy, the
diffusion coefficient of a series of labeled proteins in a wide range of
molecular mass (43-670 kD) was determined and shown to be correct with respect
to published values and the theory. Fluorescence correlation spectroscopy was
then applied to the study of fluorescently labeled tubulin and its
oligomerization in vitro induced by Mg2+ ions, paclitaxel, and a fluorescent
derivative of paclitaxel (Flutax2). By applying relations derived from the
theory of Oosawa, we were able to determine the association constant of the
oligomers induced by Mg2+. With Flutax2 our experiments show that at nanomolar
concentration, the fluorescent derivative is able to recruit tubulin dimers and
to form oligomers of defined size. Flutax2 does not bind to microtubules
preformed with paclitaxel, but it becomes preferentially incorporated into
microtubules when Flutax2 oligomers are preformed, and microtubule formation is
induced by paclitaxel addition. This shows that their incorporation into
microtubules is faster than the displacement of the prebound drug. Experiments
using fluorescently labeled tubulin and (unlabeled) paclitaxel confirm the
induction of tubulin oligomers at limiting paclitaxel concentrations.
Application of
fluorescence correlation spectroscopy for velocity imaging in microfluidic
devices. Kuricheti,
K.K., Buschmann, V., and Weston, K.D., Appl Spectrosc, 2004. 58(10):
pp. 1180-6. In this paper we present and demonstrate a technique for mapping
fluid flow rates in microfluidic systems with sub-micrometer resolution using
confocal microscopy in conjunction with fluorescence correlation spectroscopy
(FCS). Flow velocities ranging from approximately 50 microm/s to approximately
10 cm/s can be recorded using fluorescent polymer nanospheres as fluid motion
tracers. Velocity profiles and images of the flow in
poly(dimethylsiloxane)-glass microchannels are presented and analyzed. Using
the method, velocity images along the horizontal (top view) and vertical planes
within a microdevice can be obtained. This is, to our knowledge, the first
report of FCS for producing velocity maps. The high-resolution velocity maps
can be used to characterize and optimize microdevice performance and to
validate simulation efforts.
The structural
transition of the prion protein into its pathogenic conformation is induced by
unmasking hydrophobic sites. Leffers, K.W., Schell, J., Jansen, K., Lucassen, R., Kaimann, T.,
Nagel-Steger, L., Tatzelt, J., and Riesner, D., J Mol Biol, 2004. 344(3):
pp. 839-53. A series of structural intermediates in the putative pathway from
the cellular prion protein PrP(C) to the pathogenic form PrP(Sc) was
established by systematic variation of low concentrations (<0.1%) of the
detergent sodium dodecyl sulfate (SDS) or by the interaction with the bacterial
chaperonin GroEL. Most extended studies were carried out with recombinant PrP
(90-231) corresponding to the amino acid sequence of hamster prions PrP 27-30.
Similar results were obtained with full-length recombinant PrP, hamster PrP
27-30 and PrP(C) isolated from transgenic, non-infected CHO cells. Varying the
incubation conditions, i.e. the concentration of SDS, the GroEL and GroEL/ES,
but always at neutral pH and room temperature, different conformations could be
established. The conformations were characterized with respect to secondary
structure as determined by CD spectroscopy and to molecular mass, as determined
by fluorescence correlation spectroscopy and analytical ultracentrifugation:
alpha-helical monomers, soluble alpha-helical dimers, soluble but
beta-structured oligomers of a minimal size of 12-14 PrP molecules, and
insoluble multimers were observed. A high activation barrier was found between
the alpha-helical dimers and beta-structured oligomers. The numbers of
SDS-molecules bound to PrP in different conformations were determined:
Partially denatured, alpha-helical monomers bind 31 SDS molecules per PrP
molecule, alpha-helical dimers 21, beta-structured oligomers 19-20, and
beta-structured multimers show very strong binding of five SDS molecules per
PrP molecule. Binding of only five molecules of SDS per molecule of PrP leads
to fast formation of beta-structures followed by irreversible aggregation. It is
discussed that strongest binding of SDS has an effect identical with or similar
to the interaction with GroEL thereby inducing identical or very similar
transitions. The interaction with GroEL/ES stabilizes the soluble,
alpha-helical conformation. The structure and their stabilities and
particularly the induction of transitions by interaction of hydrophobic sites
of PrP are discussed in respect to their biological relevance.
Self-diffusion of
rodlike and spherical particles in a matrix of charged colloidal spheres: A
comparison between fluorescence recovery after photobleaching and fluorescence
correlation spectroscopy. Lellig, C., Wagner, J., Hempelmann, R., Keller, S., Lumma, D., and
Hartl, W., J Chem Phys, 2004. 121(14): pp. 7022-9. The
fluorescence recovery after photobleaching (FRAP) method and the fluorescence
correlation spectroscopy (FCS) have been applied on suspensions of highly
charged colloidal spheres with a small content of rod-shaped tobacco mosaic
virus (TMV) particles. Since these methods only determine the self-diffusion
coefficient of the fluorescently labeled species, D(S) of the rods and the
spheres could independently be measured. The ionic strength of the dispersion
medium has been varied to measure self-diffusion of rods and spheres in
dependence on the degree of order of the matrix spheres. In contrast to FRAP,
which allows the determination of the long-time self-diffusion coefficient D(S)
(L), FCS measures self-diffusion on a shorter time scale. Thus a comparison of
the results that were obtained by FCS and FRAP, in combination with Brownian
Dynamics simulations, gives insight into the time dependence of the
self-diffusion coefficient of an interacting colloidal system. As the mean
interparticle distance of the matrix is of the same order of magnitude as the
length of a TMV rod, the rotational motion is influenced by the assembly of
spheres around a TMV particle. Since FCS is sensitive both to translational and
rotational motion, whereas FRAP, which probes the diffusion at much larger length
scales, is only sensitive to the translational motion of TMV, the comparison of
diffusion coefficients measured employing FRAP and FCS can give some insights
in the rotational diffusion: the experimental data indicate a slowing down of
the rotational motion of a TMV rod with increasing structural order of the
matrix spheres.
Fluorescence
correlation spectroscopy and quantitative cell biology. Levin, M.K. and Carson, J.H., Differentiation,
2004. 72(1): pp. 1-10. Fluorescence correlation spectroscopy (FCS)
analyzes fluctuations in fluorescence within a small observation volume.
Autocorrelation analysis of FCS fluctuation data can be used to measure
concentrations, diffusion properties, and kinetic constants for individual
fluorescent molecules. Photon count histogram analysis of fluorescence
fluctuation data can be used to study oligomerization of individual fluorescent
molecules. If the FCS observation volume is positioned inside a living cell,
these parameters can be measured in vivo. FCS can provide the requisite
quantitative data for analysis of molecular interaction networks underlying
complex cell biological processes.
Measuring
single-molecule nucleic acid dynamics in solution by two-color filtered
ratiometric fluorescence correlation spectroscopy. Li, H., Ren, X., Ying, L.,
Balasubramanian, S., and Klenerman, D., Proc Natl Acad Sci U S A, 2004. 101(40):
pp. 14425-30. This work presents a general method for determining
single-molecule intramolecular dynamics in biomolecules by using a reporter
fluorophore, whose fluorescence is quenched or partially quenched as a result
of intramolecular motion, and a remote observer fluorophore. These fluorophores
were excited independently with two different lasers, and the ratio of the two
fluorophores' fluorescence was calculated. The time-varying ratio was then
filtered to reduce contributions from molecules outside the overlapped laser
volume and then correlated. The rates of opening and closing of a DNA hairpin
were measured by using both fluorescence correlation spectroscopy and this
method for comparison. We found at 50 pM, where molecules were studied one by
one as they diffused through the probe volume, we obtained accurate opening and
closing rates and could also measure dynamic heterogeneity. To demonstrate
applicability to a more complex biological molecule we then probed
intramolecular motions in the dimer of a human telomerase RNA fragment
(hTR(380-444)), in the presence of an excess of monomer. The motion was found
to occur on the time scale of 180-750 micros and slowed with increasing
magnesium ion concentration. Blocking experiments using complementary
oligonucleotides suggested that the motion involves substantial changes in
dimer tertiary structure. This method appears to be a general method for selectively
studying intramolecular motion in large biomolecules or complexes.
Perturbation of
Lipopolysaccharide (LPS) Micelles by Sushi 3 (S3) antimicrobial peptide. The
importance of an intermolecular disulfide bond in S3 dimer for binding,
disruption, and neutralization of LPS. Li, P., Wohland, T., Ho, B., and Ding, J.L., J Biol
Chem, 2004. 279(48): pp. 50150-6. S3 peptide, derived from the Sushi
3 domain of Factor C, which is the lipopolysaccharide (LPS)-sensitive serine
protease of the horseshoe crab coagulation cascade, was shown previously to
harbor antimicrobial activity against Gram-negative bacteria. However, the
mechanism of action remains poorly understood at the molecular level. Here we
demonstrate that the intermolecular disulfide bonding of S3 resulting in S3
dimers is indispensable for its interaction with LPS. The binding properties of
the S3 monomer and dimer to LPS were analyzed by several approaches including
enzyme-linked immunosorbent assay (ELISA)-based assay, surface plasmon
resonance, and fluorescence correlation spectroscopy (FCS). It is evident that
the S3 dimer exhibits stronger binding to LPS, demonstrating 50%
LPS-neutralizing capability at a concentration of 1 mum. Circular dichroism
spectrometry revealed that the S3 peptide undergoes conformational change in
the presence of a disulfide bridge, transitioning from a random coil to
beta-sheet structure. Using a fluorescence correlation spectroscopy monitoring
system, we describe a novel approach for examining the mechanism of peptide interaction
with LPS in the native environment. The strategy shows that intermolecular
disulfide bonding of S3 into dimers plays a critical role in its propensity to
disrupt LPS micelles and consequently neutralize LPS activity. S3 dimers
display detergent-like properties in disrupting LPS micelles. Considering
intermolecular disulfide bonds as an important parameter in the
structure-activity relationship, this insight provides clues for the future
design of improved LPS-binding and -neutralizing peptides.
Total internal
reflection with fluorescence correlation spectroscopy: nonfluorescent
competitors. Lieto,
A.M. and Thompson, N.L., Biophys J, 2004. 87(2): pp. 1268-78.
Total internal reflection with fluorescence correlation spectroscopy is a method
for measuring the surface association/dissociation rate constants and absolute
densities of fluorescent molecules at the interface of a planar substrate and
solution. This method can also report the apparent diffusion coefficient and
absolute concentration of fluorescent molecules very close to the surface.
Theoretical expressions for the fluorescence fluctuation autocorrelation
function when both surface association/dissociation kinetics and diffusion
through the evanescent wave, in solution, contribute to the fluorescence
fluctuations have been published previously. In the work described here, the
nature of the autocorrelation function when both surface
association/dissociation kinetics and diffusion through the evanescent wave
contribute to the fluorescence fluctuations, and when fluorescent and
nonfluorescent molecules compete for surface binding sites, is described. The
autocorrelation function depends in general on the kinetic association and
dissociation rate constants of the fluorescent and nonfluorescent molecules,
the surface site density, the concentrations of fluorescent and nonfluorescent
molecules in solution, the solution diffusion coefficients of the two chemical
species, the depth of the evanescent field, and the size of the observed area
on the surface. Both general and approximate expressions are presented.
Technological
advances in high-throughput screening. Liu, B., Li, S., and Hu, J., Am J Pharmacogenomics,
2004. 4(4): pp. 263-76. High-throughput screening (HTS) is the process
of testing a large number of diverse chemical structures against disease
targets to identify 'hits'. Compared to traditional drug screening methods, HTS
is characterized by its simplicity, rapidness, low cost, and high efficiency,
taking the ligand-target interactions as the principle, as well as leading to a
higher information harvest. As a multidisciplinary field, HTS involves an
automated operation-platform, highly sensitive testing system, specific
screening model (in vitro), an abundant components library, and a data
acquisition and processing system. Various technologies, especially the novel
technologies such as fluorescence, nuclear-magnetic resonance, affinity
chromatography, surface plasmon resonance, and DNA microarray, are now
available, and the screening of more than 100,000 samples per day is already
possible. Fluorescence-based assays include the scintillation proximity assay,
time-resolved energy transfer, fluorescence anisotropy, fluorescence
correlation spectroscopy, and fluorescence fluctuation spectroscopy.
Fluorescence-based techniques are likely to be among the most important
detection approaches used for HTS due to their high sensitivity and amenability
to automation, giving the industry-wide drive to simplify, miniaturize, and
speed up assays. The application of NMR technology to HTS is another recent
trend in drug research. One advantage afforded by NMR technology is that it can
provide direct information on the affinity of the screening compounds and the
binding location of protein. The structure-activity relationship acquired from
NMR analysis can sharpen the library design, which will be very important in
furnishing HTS with well-defined drug candidates. Affinity chromatography used
for library screening will provide the information on the fundamental processes
of drug action, such as absorption, distribution, excretion, and receptor
activation; also the eluting curve can give directly the possibility of
candidate drug. SPR can measure the quantity of a complex formed between two
molecules in real-time without the need for fluorescent or radioisotopic
labels. SPR is capable of characterizing unmodified biopharmaceuticals,
studying the interaction of drug candidates with macromolecular targets, and
identifying binding partners during ligand fishing experiments. DNA microarrays
can be used in HTS be used to further investigate the expression of biological
targets associated with human disease, which then opens new and exciting
opportunities for drug discovery. Without doubt, the addition of new
technologies will further increase the application of HTS in drug screening and
its related fields.
Structure property
analysis of pentamethine indocyanine dyes: identification of a new dye for life
science applications.
Mader, O., Reiner, K., Egelhaaf, H.J., Fischer, R., and Brock, R., Bioconjug
Chem, 2004. 15(1): pp. 70-8. A collection of nine pentamethine
indocyanine dyes was synthesized, and the photophysical characteristics
relevant to applications in cell biology and single molecule detection were analyzed
in detail. Substituents at the aromatic system covering the auxochromic series
and substitutions in the polymethine chain were investigated with respect to
absorption and emission spectra, fluorescence lifetimes, fluorescence quantum
yields, and fluorescence autocorrelations. Substitutions in the polymethine
chain increased the nonradiative energy dissipation of the excited singlet
state and decreased the fluorescence quantum yield, relative to the
unsubstituted compound. For substituents at the aromatic rings the fluorescence
quantum yield negatively correlates with the position of the substituents in
the auxochromic series -SO(3)(-), -H, -F, -CH(3). Compounds with sulfonic acid
groups or halogen atoms attached to the indolenine systems had the highest
fluorescence quantum yields. The compound S0387 had nearly 70% of the quantum
yield of Cy5 and comparable photostability. The free carboxylic acid of S0387
was attached to peptides in high yield and purity by established procedures of
solid-phase synthesis. The dye-labeled peptides did not aggregate or bind to
tissue culture cells and proteins unspecifically. The indocyanine dye S0387 is
therefore an attractive new fluorophore for in vitro and cell-based detection
of receptor ligand interaction at nanomolar concentrations by flow cytometry,
fluorescence correlation spectroscopy, and laser scanning microscopy.
Fluorescence
correlation spectroscopy: incorporation of probe volume effects into the
three-dimensional Gaussian approximation. Marrocco, M., Appl Opt, 2004. 43(27): pp.
5251-62. Fluorescence correlation spectroscopy is a valuable tool in many
scientific disciplines. In particular, such a spectroscopic technique has
received a great deal of attention because of its remarkable potential for
single-molecule detection. It is understood, however, that quantitative
measurements can be considered reliable as long as molecular photophysics has
been well characterized. To that end, molecular saturation and probe volume
effects, which can worsen experimental accuracy, are treated here. These
phenomena are adequately incorporated into the well-known three-dimensional
Gaussian approximation by a novel method applied to interpret saturated
fluorescence signals [Opt. Lett. 28, 2016 (2003)]. Comparisons with literature
data are given to show the improvements of the suggested method compared with
other approaches.
A new approach for
fluorescence correlation spectroscopy (FCS) based immunoassays. Mayboroda, O.A., van Remoortere,
A., Tanke, H.J., Hokke, C.H., and Deelder, A.M., J Biotechnol, 2004. 107(2):
pp. 185-92. Fluorescence correlation spectroscopy (FCS) is a powerful technique
for measuring physicochemical properties, such as concentration and diffusion
constant, of bio-molecules in complex mixtures. Although, as such, FCS is well
suited for development of homogeneous immunoassays, a major obstacle lies in
the relatively high molecular weight of antibodies. This is because in FCS
discrimination between unbound fluorescently-labelled antibodies and the same
antibodies bound to immune complexes is based on the difference of their
respective diffusion coefficients. To overcome this limitation we here propose
to use a fluorescently-labelled tag which has two crucial properties: (a) its
molecular weight is significantly lower than that of an antibody and (b) it is
capable to discriminate between free antibodies and immune complexes. We have
evaluated the feasibility of this approach in a model system consisting of
mouse monoclonal IgG directed against the Lewis X antigen, and Protein A as a
low molecular weight tag.
Modifying the
adsorption behavior of polyamidoamine dendrimers at silica surfaces
investigated by total internal reflection fluorescence correlation spectroscopy. McCain, K.S., Schluesche, P., and
Harris, J.M., Anal Chem, 2004. 76(4): pp. 930-8. Polyamidoamine
(PAMAM) dendrimers were modified and tested for use as solution-phase diffusion
probes in silica nanostructures. In order for the successful application of
dendrimers as solution-phase probes, their interactions with silica surfaces
must be understood and controlled, so that the motion of the probe is not
influenced by adsorption. Adsorption/desorption kinetics of PAMAM dendrimers
and their diffusion in solution near silica surfaces were investigated with total
internal reflection fluorescence correlation spectroscopy (TIR-FCS). Dendrimers
of generations 3, 5, and 7 were dye-labeled with carboxyrhodamine 6G. Because
PAMAM dendrimers are positively charged in solution (having primary amines as
end groups), significant adsorption of these molecules to the negatively
charged silica surface was observed. Adsorption/desorption rates and the
equilibrium constant for adsorption were determined by fitting the
autocorrelation functions to a kinetic model. The desorption rate decreases and
the absorption equilibrium constant increases with higher dendrimer generation.
To reduce the adsorption of these probes to silica surfaces, the labeled
dendrimers were reacted with succinic anhydride, converting the primary amine
end groups to negatively charged carboxylic acid groups. These carboxylated
dendrimers did not detectably adsorb to silica from aqueous solution. TIR-FCS
was used to determine their free-solution diffusion constants near silica
surfaces, and the corresponding hydrodynamic radii compare favorably with
values reported from forced Rayleigh scattering measurements.
Structure and
dynamics of lipoplex formation examined using two-photon fluorescence
cross-correlation spectroscopy. Merkle, D., Lees-Miller, S.P., and Cramb, D.T., Biochemistry,
2004. 43(23): pp. 7263-72. The conditions required to form transfectable
lipoplexes have been extensively studied [Zuhorn, I. S., and Hoekstra, D.
(2002) J. Membr. Biol. 189, 167-179]. However, to date, experiments have not
addressed either the order of events of lipoplex formation in solution or the
maximum number of DNA molecules per vesicle in stable single-vesicle
lipoplexes. In this study, we have employed two-photon excitation fluorescence
correlation spectroscopy (TPE-FCS) and two-photon fluorescence
cross-correlation spectroscopy (TPE-XCS) to examine both fluorescence-labeled
DNA and cationic vesicle structure and dynamics simultaneously. The dependence
of large aggregated lipoplex formation on DNA-to-cationic lipid charge ratio
was determined, as was the maximum number of 40 bp double-stranded DNA
oligonucleotides able to bind to a single vesicle.
Interaction assay of
oligosaccharide with lectins using glycosylasparagine. Mizuno, M., Noguchi, M., Imai, M.,
Motoyoshi, T., and Inazu, T., Bioorg Med Chem Lett, 2004. 14(2):
pp. 485-90. Glycosyl amino acids having natural glycan were useful for the
interaction assay of oligosaccharides. A glycochip containing the whole
structure of an oligosaccharide was easily prepared by the immobilization of
the glycosyl amino acid. Furthermore, fluorescence probes were introduced into
the glycosyl amino acid while maintaining the whole structure of
oligosaccharide. By using these labeled oligosaccharides, fluorescence
polarization (FP) and fluorescence correlation spectroscopy (FCS) analyzed the
carbohydrate-lectin interaction in a solution assay system.
Maximum-entropy
decomposition of fluorescence correlation spectroscopy data: application to
liposome-human serum albumin association. Modos, K., Galantai, R., Bardos-Nagy, I., Wachsmuth, M.,
Toth, K., Fidy, J., and Langowski, J., Eur Biophys J, 2004. 33(1):
pp. 59-67. Fluorescence correlation spectroscopy was used to measure the
diffusion behavior of a mixture of DMPC or DMPC/DMPG liposomes with human serum
albumin (HSA) and mesoporphyrin (MP), which was used as the fluorescent label
for liposomes and HSA as well. For decomposing the fluorescence intensity
autocorrelation function (ACF) into components corresponding to a liposome
population, HSA and MP, we used a maximum entropy procedure that computes a
distribution of diffusion times consistent with the ACF data. We found that a
simple parametric non-linear fit with a discrete set of decay components did not
converge to a stable parameter set. The distribution calculated with the
maximum entropy method was stable and the average size of the particles
calculated from the effective diffusion time was in good agreement with the
data determined using the discrete-component fit.
How confined
lubricants diffuse during shear. Mukhopadhyay, A., Bae, S.C., Zhao, J., and Granick, S., Phys
Rev Lett, 2004. 93(23): p. 236105. The translational diffusion of a
fluorescent dye embedded at a dilute concentration in a confined fluid was
compared at rest and during shear. The fluid, octamethylcyclotetrasiloxane
(OMCTS), was confined between step-free muscovite mica to thickness 3-4 layers.
Fluorescence correlation spectroscopy showed that the time scales of
intensity-intensity autocorrelation functions were essentially the same during
shear and at rest, except they were faster during shear by a factor of 2 to 5.
This dynamical probe of how liquids order in molecularly thin films fails to
support the hypothesis that shear produced a melting transition.
Two-dimensional
fluorescence correlation spectroscopy II: spectral analysis of derivatives of
anthracene and pyrene in micellar solutions. Nakashima, K., Yuda, K., Ozaki, Y., and Noda, I., Spectrochim
Acta A Mol Biomol Spectrosc, 2004. 60(8-9): pp. 1783-91. Generalized
two-dimensional (2D) correlation spectroscopy has been applied to the analysis
of fluorescence spectra in two micellar systems: (1) a mixture of pyrene and
1,3,6,8-pyrenetetrasulfonic acid in the cationic micellar solutions of
cetyltrimethylammonium chloride (CTAC) and (2) a mixture of pyrene and
9-anthracencepropionic acid in anionic micellar solutions of sodium dodecyl
sulfate (SDS). Fluorescence quenching is employed as a perturbation mode for
causing intensity changes in fluorescence bands (quenching perturbation).
Iodide ion (I-) is used as a quencher in the former system, and cetyl pridinium
chloride (CPC) is used in the latter. Vibronic bands in the complicated
fluorescence spectra of the mixture of the analytes were successfully resolved.
It is shown that asynchronous maps are especially useful for spectral
resolution enhancement when the quenching perturbation is employed in 2D
fluorescence correlation spectroscopy. Furthermore, the information about the
order of response of the bands to quenching is obtained by comparing the signs
of synchronous and asynchronous cross-peaks.
Photophysical aspects
of single-molecule detection by two-photon excitation with consideration of
sequential pulsed illumination. Niesner, R., Roth, W., and Gericke, K.H., Chemphyschem,
2004. 5(5): pp. 678-87. An important goal in single molecule
fluorescence correlation spectroscopy is the theoretical simulation of the
fluorescence signal stemming from individual molecules and its autocorrelation
function. The simulation approaches developed up to now are based exclusively
on continuous-wave (cw) illumination and consequently on cw-excitation.
However, this approximation is no longer valid in the case of two-photon
excitation, for which pulsed illumination is usually employed. We present a
novel theoretical model for the simulation of the fluorescence signal of single
molecules and its autocorrelation function with consideration of the time
dependence of the excitation flux and thus of all illumination-dependent
photoprocesses: two-photon excitation, induced emission and photobleaching.
Further important characteristics of our approach are the consideration of the
dependence of the photobleaching rate on illumination and the low
intersystem-crossing rates of the studied coumarins. Moreover, using our
approach, we can predict quantitatively the effect of the laser pulse width on
the fluorescence signal of a molecule, that is, the contributions of the
photobleaching and saturation effects, and thus we can calculate the optimal
laser pulse width. The theoretical autocorrelation functions were fitted to the
experimental data, and we could ascertain a good agreement between the
resulting and the expected parameters. The most important parameter is the photobleaching
constant sigma, the cross section of the transition Sn<--S1, which
characterises the photostability of the molecules independent of the
experimental conditions. Its value is 1.7 x 10(-23) cm2 for coumarin 153 and 5
x 10(-23) cm2 for coumarin 314.
Real-time monitoring
of in vitro transcriptional RNA by using fluorescence correlation spectroscopy. Nomura, Y. and Kinjo, M., Chembiochem,
2004. 5(12): pp. 1701-3.
Vesicle encapsulation
studies reveal that single molecule ribozyme heterogeneities are intrinsic. Okumus, B., Wilson, T.J., Lilley,
D.M., and Ha, T., Biophys J, 2004. 87(4): pp. 2798-806.
Single-molecule measurements have revealed that what were assumed to be
identical molecules can differ significantly in their static and dynamic
properties. One of the most striking examples is the hairpin ribozyme, which
was shown to exhibit two to three orders of magnitude variation in folding
kinetics between molecules. Although averaged behavior of single molecules
matched the bulk solution data, it was not possible to exclude rigorously the
possibility that the variations around the mean values arose from different
ways of interacting with the surface environment. To test this, we minimized
the molecules' interaction with the surface by encapsulating DNA or RNA
molecules inside 100- to 200-nm diameter unilamellar vesicles, following the
procedures described by Haran and coworkers. Vesicles were immobilized on a
supported lipid bilayer via biotin-streptavidin linkages. We observed no direct
binding of DNA or RNA on the supported bilayer even at concentrations exceeding
100 nM, indicating that these molecules do not bind stably on the membrane.
Since the vesicle diameter is smaller than the resolution of optical
microscopy, the lateral mobility of the molecules is severely constrained,
allowing long observation periods. We used fluorescence correlation
spectroscopy, nuclease digestion, and external buffer exchange to show that the
molecules were indeed encapsulated within the vesicles. When contained within vesicles,
the natural form of the hairpin ribozyme exhibited 50-fold variation in both
folding and unfolding rates in 0.5 mM Mg2+, which is identical to what was
observed from the molecules tethered directly on the surface. This strongly
indicates that the observed heterogeneity in dynamic properties does not arise
as an artifact of surface attachment, but is intrinsic to the nature of the
molecules.
New chimera proteins
for fluorescence correlation spectroscopy. Olah, Z., Trier, U., Sauer, B., Schafer-Korting,
M., and Kleuser, B., Pharmazie, 2004. 59(7): pp. 516-23. A new
class of chimera proteins has been developed. They are ideally suited for
detection by fluorescence correlation spectroscopy (FCS), a new technology to
analyze molecular interactions. The molecular structure of these chimera
proteins consists of four domains: a N-terminal (His)6-tag for affinity
chromatography followed by an eight amino acid epitope for immunodetection, a
polypeptide affinity domain (ADF) for target specific interaction and a
C-terminal Green Fluorescent Protein (GFPuv) for reporting of interaction with
the target by FCS. We designed, prepared and characterized a prototype of
ADF-GFP proteins capable of specific interaction with DNA fragments bearing
nuclear factor (NF)-kappaB sites. ADF NF-kappaB p50 and a non-DNA-binding
deletion mutant (p35) combined with GFPuv were inserted in a procaryotic vector
and expressed in E. coli. Following affinity purification the fluoroproteins
p50-GFPuv and p35-GFPuv were employed in specific protein-protein and
protein-DNA interaction studies. FCS analysis as well as EMSA showed that
p50-GFPuv revealed a fully functional ADF. We present a model for the
preparation of GFP fusion proteins capable of specific interaction with
proteins, lipids or nucleic acids. The rational design allows any polypeptide
fragment to be incorporated into the chimeric protein. So a new series of
bio-molecules with different binding specificities and assays can be developed.
Ligand-receptor interactions
in live cells by fluorescence correlation spectroscopy. Pramanik, A., Curr Pharm
Biotechnol, 2004. 5(2): pp. 205-12. Receptor binding studies most
often require the use of radioactively labeled ligands. In certain cases, the
numbers of receptors are few per cell and no specific binding is detected
because of a high background. Specific interactions between certain ligands
(e.g. peptides, hormones, natural products) and their receptors are, therefore,
often overlooked by the conventional binding technique. Fluorescence
correlation spectroscopy (FCS) allows detection of the interaction of ligands
with receptors in their native environment in live cells in a tiny confocal
volume element (0.2 fl) at single-molecule detection sensitivity. This technique
permits the identification of receptors which were not possible before to
detect by isotope labeling. The beauty of the FCS technique is that there is no
need for separating an unbound ligand from a bound one to calculate the
receptor bound and free ligand fractions. This review will show FCS as a
sensitive and a rapid technique to study ligand-receptor interaction in live
cells and will demonstrate that the FCS-analysis of ligand-receptor
interactions in live cells fulfils all the criteria of a ligand binding to its
receptor i.e. it is able to provide information on the affinity and specificity
of a ligand, binding constant, association and dissociation rate constants as
well as the number and mobility of receptors carrying a fluorescently labeled
ligand. This review is of pharmaceutical significance since it will provide
insights on how FCS can be used as a rapid technique for studying
ligand-receptor interactions in cell cultures, which is one step forward
towards a high throughput drug screening in cell cultures.
Determination of the
bioavailability of biotin conjugated onto shell cross-linked (SCK)
nanoparticles. Qi,
K., Ma, Q., Remsen, E.E., Clark, C.G., Jr., and Wooley, K.L., J Am Chem Soc,
2004. 126(21): pp. 6599-607. Shell cross-linked nanoparticles (SCKs)
presenting surface- and bioavailable biotin functional groups were synthesized
via a mixed micelle methodology, whereby co-micellization of chain terminal
biotinylated poly(acrylic acid)-b-poly(methyl acrylate) (PAA-b-PMA) and
nonbiotinylated PAA-b-PMA were cross-linked in an intramicellar fashion within
the shell layer of the mixed micelles, between the carboxylic acid groups of
PAA and the amine functionalities of 2,2'-(ethylenedioxy)diethylamine. The
hydrodynamic diameters (D(h)) of the micelles and the SCKs with different
biotinylated block copolymer contents were determined by dynamic light
scattering (DLS), and the dimensions of the SCKs were characterized with
tapping-mode atomic force microscopy (AFM) and transmission electron microscopy
(TEM). The amount of surface-available biotin was tuned by varying the
stoichiometric ratio of the biotinylated PAA-b-PMA versus the nonbiotinylated
PAA-b-PMA, as demonstrated with solution-state, binding interaction analyses,
an avidin/HABA (avidin/4'-hydroxyazobenzene-2-carboxylic acid) competitive
binding assay, and fluorescence correlation spectroscopy (FCS). The avidin/HABA
assay found the amount of available biotin at the surface of the biotinylated
SCK nanoparticles to increase with increasing biotin-terminated block copolymer
incorporation, but to be less than 25% of the theoretical value. FCS
measurements showed the same trend.
Fluorescence
correlation spectroscopy with high-order and dual-color correlation to probe
nonequilibrium steady states. Qian, H. and Elson, E.L., Proc Natl Acad Sci U S A,
2004. 101(9): pp. 2828-33. In living cells, biochemical reaction
networks often function in nonequilibrium steady states. Under these
conditions, the networks necessarily have cyclic reaction kinetics that are
maintained by sustained constant input and output, i.e., pumping. To
differentiate this state from an equilibrium state without flux, we propose a
microscopic method based on concentration fluctuation measurements, via
fluorescence correlation spectroscopy, and statistical analyses of high-order
correlations and cross correlations beyond the standard fluorescence
correlation spectroscopy autocorrelation. We show that, for equilibrium systems
with time reversibility, the correlation functions possess certain symmetries,
the violation of which is a measure of steady-state fluxes in reaction cycles.
This result demonstrates the theoretical basis for experimentally measuring
reaction fluxes in a biochemical network in situ and the importance of
single-molecule measurements in providing fundamental information on
nonequilibrium steady-states in biochemistry.
FRET-FCS as a tool to
evaluate the stability of oligonucleotide drugs after intracellular delivery. Remaut, K., Lucas, B., Braeckmans,
K., Sanders, N.N., De Smedt, S.C., and Demeester, J., J Control Release,
2005. 103(1): pp. 259-71. The intracellular degradation of
single-stranded, double-labeled oligonucleotides (ONs) was studied by following
the disappearance of Fluorescence Resonance Energy Transfer (FRET) between the
rhodamine green and Cy5 fluorophores attached to respectively the 3' and 5' end
of the ONs. The green and red fluorescence intensities upon rhodamine green
excitation were monitored using the ultra-sensitive detectors of a dual-color
Fluorescence Correlation Spectroscopy (FCS) instrument. The ratio of the red to
green fluorescence (R/G ratio) as obtained from such FRET-FCS measurements
showed to give accurate information on the integrity of the ONs, without the
need for additional auto- or cross-correlation analysis of the registered
fluorescence intensity fluctuations. Intracellular measurements revealed that
most of the 40mer phosphodiester ONs were degraded before they entered the
nucleus. For the 20mer phosphodiester ONs, this degradation occurred more
slowly, and both intact and degraded ONs entered the nucleus. For the 20mer
phosphorothioate ONs, no intracellular degradation was observed during the
measured time period. The sensitive detection of the intracellular fluorescence
by the FCS setup will be particularly useful in situations where the expected
fluorescence is too low to be detected by FRET-imaging as may occur after
intracellular delivery of ONs by cationic carriers.
Discerning
aggregation in homogeneous ensembles: a general description of photon counting
spectroscopy in diffusing systems. Ren, H.C., Goddard, N.L., Altan-Bonnet, G., and Libchaber,
A., Phys Rev E Stat Nonlin Soft Matter Phys, 2004. 69(5 Pt 1): p.
051916. In order to discern aggregation in solution, we present a quantum
mechanical analog of the photon statistics from fluorescent molecules diffusing
through a focused beam. A generating functional is developed to fully describe
the experimental physical system as well as the statistics. Histograms of the
measured time delay between photon counts are fit by an analytical solution
describing the static as well as diffusing regimes. To determine empirical
fitting parameters, fluorescence correlation spectroscopy is used in parallel
to the photon counting. For expedient analysis, we find that the distribution's
deviation from a single Poisson shows a difference between two single fluor
monomers or a double fluor aggregate of the same total intensities. Initial
studies were performed on fixed-state aggregates limited to dimerization.
However preliminary results on reactive species suggest that the method can be
used to characterize any aggregating system.
pH microdomains in
oligodendrocytes.
Ro, H.A. and Carson, J.H., J Biol Chem, 2004. 279(35): pp.
37115-23. Oligodendrocytes (OLs) are cells that produce myelin in the central
nervous system. Here we use ratiometric pH indicator dye to analyze
intracellular pH in OLs in culture. The results reveal alkaline microdomains,
which predominate in the perikaryon and proximal dendrites, and acidic
microdomains, which predominate in distal dendrites. Spatial nonuniformity of
pH is generated by differential subcellular distribution of Na(+)/H(+)
exchanger (NHE), which is localized in a punctate distribution in the
perikaryon and proximal processes, Na(+)/HCO(3)(-) cotransporter (NBC), which
is localized in a punctate distribution in distal dendrites, and carbonic
anhydrase isotype II (CAII), which is colocalized with either NHE or NBC.
Inhibition of NHE activity by amiloride inhibits regeneration of alkaline
microdomains after cytoplasmic acidification, whereas the inhibition of CAII
activity with ethoxyzolamide inhibits acidification of dendrites. Fluorescence
correlation spectroscopy analysis of CAII microinjected into OLs reveals freely
diffusing protein throughout the cell as well as protein associated
predominantly with NHE in the perikaryon and predominantly with NBC in the
dendrites. Alkaline and acidic microdomains could be generated by transport
metabolons consisting of CAII associated with NHE or NBC, respectively. This
study provides the first evidence for pH microdomains in cells and describes a
mechanism for how they are generated.
Spatial-temporal
studies of membrane dynamics: scanning fluorescence correlation spectroscopy
(SFCS). Ruan, Q.,
Cheng, M.A., Levi, M., Gratton, E., and Mantulin, W.W., Biophys J, 2004.
87(2): pp. 1260-7. Giant unilamellar vesicles (GUVs) have been widely
used as a model membrane system to study membrane organization, dynamics, and
protein-membrane interactions. Most recent studies have relied on imaging
methods, which require good contrast for image resolution. Multiple sequential
image processing only detects slow components of membrane dynamics. We have
developed a new fluorescence correlation spectroscopy (FCS) technique, termed
scanning FCS (i.e., SFCS), which performs multiple FCS measurements
simultaneously by rapidly directing the excitation laser beam in a uniform
(circular) scan across the bilayer of the GUVs in a repetitive fashion. The
scan rate is fast compared to the diffusion of the membrane proteins and even
small molecules in the GUVs. Scanning FCS outputs a "carpet" of timed
fluorescence intensity fluctuations at specific points along the scan. In this
study, GUVs were assembled from rat kidney brush border membranes, which
included the integral membrane proteins. Scanning FCS measurements on GUVs
allowed for a straightforward detection of spatial-temporal interactions
between the protein and the membrane based on the diffusion rate of the protein.
To test for protein incorporation into the bilayers of the GUVs, antibodies
against one specific membrane protein (NaPi II cotransporter) were labeled with
ALEXA-488. Fluorescence images of the GUVs in the presence of the labeled
antibody showed marginal fluorescence enhancement on the GUV membrane bilayers
(poor image contrast and resolution). With the application of scanning FCS, the
binding of the antibody to the GUVs was detected directly from the analysis of
diffusion rates of the fluorescent antibody. The diffusion coefficient of the
antibody bound to NaPi II in the GUVs was approximately 200-fold smaller than
that in solution. Scanning FCS provided a simple, quantitative, yet highly
sensitive method to study protein-membrane interactions.
Pleckstrin homology
domain diffusion in Dictyostelium cytoplasm studied using fluorescence
correlation spectroscopy. Ruchira, Hink, M.A., Bosgraaf, L., van Haastert, P.J., and Visser,
A.J., J Biol Chem, 2004. 279(11): pp. 10013-9. The translocation
of pleckstrin homology (PH) domain-containing proteins from the cytoplasm to
the plasma membrane plays an important role in the chemotaxis mechanism of
Dictyostelium cells. The diffusion of three PH domain-green fluorescent protein
(GFP) fusions (PH2-GFP, PH10-GFP, and PH-CRAC (cytosolic regulator of adenylyl
cyclase)-GFP) in the cytoplasm of vegetative and chemotaxing Dictyostelium
cells has been studied using fluorescence correlation spectroscopy to gain a
better understanding of the functioning of the domains and to assess the effect
of initiation of chemotaxis on these domains in the cell. PH2-GFP was
homogeneously distributed in vegetative as well as chemotaxing cells, whereas
PH10-GFP and PH-CRAC-GFP showed translocation to the leading edge of the
chemotaxing cell. The diffusion characteristics of PH2-GFP and PH-CRAC-GFP were
very similar; however, PH10-GFP exhibited slower diffusion. Photon counting
histogram statistics show that this slow diffusion was not due to aggregation.
Diffusion of the three PH domains was affected to similar extents by
intracellular heterogeneities in vegetative as well as chemotaxing cells. From
the diffusion of free cytoplasmic GFP, it was calculated that the viscosity in
chemotaxing cells was 1.7 times lower than in vegetative cells. In chemotaxing
cells, PH2-GFP showed increased mobility, whereas the mobilities of PH10-GFP
and PH-CRAC-GFP remained unchanged.
Fluorescence
correlation spectroscopy studies of Peptide and protein binding to phospholipid
vesicles. Rusu, L.,
Gambhir, A., McLaughlin, S., and Radler, J., Biophys J, 2004. 87(2):
pp. 1044-53. We used fluorescence correlation spectroscopy (FCS) to analyze the
binding of fluorescently labeled peptides to lipid vesicles and compared the
deduced binding constants to those obtained using other techniques. We used a
well-characterized peptide corresponding to the basic effector domain of
myristoylated alanine-rich C kinase substrate, MARCKS(151-175), that was
fluorescently labeled with Alexa488, and measured its binding to large unilamellar
vesicles (diameter approximately 100 nm) composed of phosphatidylcholine and
phosphatidylserine or phosphatidylinositol 4,5-bisphosphate. Because the large
unilamellar vesicles are significantly larger than the peptide, the correlation
times for the free and bound peptide could be distinguished using single color
autocorrelation measurements. The molar partition coefficients calculated from
the FCS measurements were comparable to those obtained from binding
measurements of radioactively labeled MARCKS(151-175) using a centrifugation
technique. Moreover, FCS can measure binding of peptides present at very low
concentrations (1-10 nmolar), which is difficult or impossible with most other
techniques. Our data indicate FCS can be an accurate and valuable tool for
studying the interaction of peptides and proteins with lipid membranes.
Prevention of
Alzheimer's disease-associated Abeta aggregation by rationally designed
nonpeptidic beta-sheet ligands. Rzepecki, P., Nagel-Steger, L., Feuerstein, S., Linne, U.,
Molt, O., Zadmard, R., Aschermann, K., Wehner, M., Schrader, T., and Riesner,
D., J Biol Chem, 2004. 279(46): pp. 47497-505. A new concept is
introduced for the rational design of beta-sheet ligands, which prevent protein
aggregation. Oligomeric acylated aminopyrazoles with a donor-acceptor-donor
(DAD) hydrogen bond pattern complementary to that of a beta-sheet efficiently
block the solvent-exposed beta-sheet portions in Abeta-(1-40) and thereby
prevent formation of insoluble protein aggregates. Density gradient
centrifugation revealed that in the initial phase, the size of Abeta aggregates
was efficiently kept between the trimeric and 15-meric state, whereas after 5
days an additional high molecular weight fraction appeared. With fluorescence
correlation spectroscopy (FCS) exactly those two, i.e. a dimeric aminopyrazole
with an oxalyl spacer and a trimeric head-to-tail connected aminopyrazole, of
nine similar aminopyrazole ligands were identified as efficient aggregation
retardants whose minimum energy conformations showed a perfect complementarity
to a beta-sheet. The concentration dependence of the inhibitory effect of a
trimeric aminopyrazole derivative allowed an estimation of the dissociation
constant in the range of 10(-5) m. Finally, electrospray ionization mass
spectrometry (ESI-MS) was used to determine the aggregation kinetics of
Abeta-(1-40) in the absence and in the presence of the ligands. From the
comparable decrease in Abeta monomer concentration, we conclude that these
beta-sheet ligands do not prevent the initial oligomerization of monomeric
Abeta but rather block further aggregation of spontaneously formed small
oligomers. Together with the results from density gradient centrifugation and
fluorescence correlation spectroscopy it is now possible to restrict the
approximate size of soluble Abeta aggregates formed in the presence of both
inhibitors from 3- to 15-mers.
lambda-Repressor
oligomerization kinetics at high concentrations using fluorescence correlation
spectroscopy in zero-mode waveguides. Samiee, K.T., Foquet, M., Guo, L., Cox, E.C., and
Craighead, H.G., Biophys J, 2005. 88(3): pp. 2145-53.
Fluorescence correlation spectroscopy (FCS) has demonstrated its utility for
measuring transport properties and kinetics at low fluorophore concentrations.
In this article, we demonstrate that simple optical nanostructures, known as
zero-mode waveguides, can be used to significantly reduce the FCS observation
volume. This, in turn, allows FCS to be applied to solutions with significantly
higher fluorophore concentrations. We derive an empirical FCS model accounting
for one-dimensional diffusion in a finite tube with a simple exponential
observation profile. This technique is used to measure the oligomerization of
the bacteriophage lambda repressor protein at micromolar concentrations. The
results agree with previous studies utilizing conventional techniques.
Additionally, we demonstrate that the zero-mode waveguides can be used to assay
biological activity by measuring changes in diffusion constant as a result of
ligand binding.
Tubulin equilibrium
unfolding followed by time-resolved fluorescence and fluorescence correlation
spectroscopy.
Sanchez, S.A., Brunet, J.E., Jameson, D.M., Lagos, R., and Monasterio, O., Protein
Sci, 2004. 13(1): pp. 81-8. The pathway for the in vitro equilibrium
unfolding of the tubulin heterodimer by guanidinium chloride (GdmCl) has been
studied using several spectroscopic techniques, specifically circular dichroism
(CD), two-photon Fluorescence Correlation Spectroscopy (FCS), and time-resolved
fluorescence, including lifetime and dynamic polarization. The results show
that tubulin unfolding is characterized by distinct processes that occur in
different GdmCl concentration ranges. From 0 to 0.5 M GdmCl, a slight
alteration of the tubulin heterodimer occurs, as evidenced by a small, but
reproducible increase in the rotational correlation time of the protein and a
sharp decrease in the secondary structure monitored by CD. In the range 0.5-1.5
M GdmCl, significant decreases in the steady-state anisotropy and average
lifetime of the intrinsic tryptophan fluorescence occur, as well as a decrease
in the rotational correlation time, from 48 to 26 nsec. In the same GdmCl
range, the number of protein molecules (labeled with Alexa 488), as determined
by two-photon FCS measurements, increases by a factor of two, indicating
dissociation of the tubulin dimer into monomers. From 1.5 to 4 M GdmCl, these
monomers unfold, as evidenced by the continual decrease in the tryptophan
steady-state anisotropy, average lifetime, and rotational correlation time,
concomitant with secondary structural changes. These results help to elucidate
the unfolding pathway of the tubulin heterodimer and demonstrate the value of
FCS measurements in studies on oligomeric protein systems.
Photodynamics of red
fluorescent proteins studied by fluorescence correlation spectroscopy. Schenk, A., Ivanchenko, S.,
Rocker, C., Wiedenmann, J., and Nienhaus, G.U., Biophys J, 2004. 86(1
Pt 1): pp. 384-94. Red fluorescent proteins are important tools in
fluorescence-based life science research. Recently, we have introduced eqFP611,
a red fluorescent protein with advantageous properties from the sea anemone
Entacmaea quadricolor. Here, we have studied the submillisecond light-driven
intramolecular dynamics between bright and dark states of eqFP611 and, for
comparison, drFP583 (DsRed) by using fluorescence correlation spectroscopy on
protein solutions. A three-state model with one dark and two fluorescent states
describes the power-dependence of the flickering dynamics of both proteins at
different excitation wavelengths. It involves two light-driven conformational
transitions. We have also studied the photodynamics of individual (monomeric)
eqFP611 molecules immobilized on surfaces. The flickering rates and dark state
fractions of eqFP611 bound to polyethylene glycol-covered glass surfaces were
identical to those measured in solution, showing that the bound FPs behaved
identically. A second, much slower flickering process was observed on the 10-ms
timescale. Deposition of eqFP611 molecules on bare glass surfaces yielded
bright fluorescence without any detectable flickering and a >10-fold
decreased photobleaching yield. These observations underscore the intimate
connection between protein motions and photophysical processes in fluorescent
proteins.
High- and
low-mobility populations of HP1 in heterochromatin of mammalian cells. Schmiedeberg, L., Weisshart, K.,
Diekmann, S., Meyer Zu Hoerste, G., and Hemmerich, P., Mol Biol Cell,
2004. 15(6): pp. 2819-33. Heterochromatin protein 1 (HP1) is a conserved
nonhistone chromosomal protein with functions in euchromatin and
heterochromatin. Here we investigated the diffusional behaviors of HP1 isoforms
in mammalian cells. Using fluorescence correlation spectroscopy (FCS) and
fluorescence recovery after photobleaching (FRAP) we found that in interphase
cells most HP1 molecules (50-80%) are highly mobile (recovery halftime: t(1/2)
approximately 0.9 s; diffusion coefficient: D approximately 0.6-0.7 microm(2)
s(-1)). Twenty to 40% of HP1 molecules appear to be incorporated into stable,
slow-moving oligomeric complexes (t(1/2) approximately 10 s), and constitutive
heterochromatin of all mammalian cell types analyzed contain 5-7% of very slow
HP1 molecules. The amount of very slow HP1 molecules correlated with the
chromatin condensation state, mounting to more than 44% in condensed chromatin
of transcriptionally silent cells. During mitosis 8-14% of GFP-HP1alpha, but
not the other isoforms, are very slow within pericentromeric heterochromatin,
indicating an isoform-specific function of HP1alpha in heterochromatin of
mitotic chromosomes. These data suggest that mobile as well as very slow
populations of HP1 may function in concert to maintain a stable conformation of
constitutive heterochromatin throughout the cell cycle.
DNA measurements by
using fluorescence correlation spectroscopy and two-color fluorescence cross
correlation spectroscopy. Takagi, T., Kii, H., and Kinjo, M., Curr Pharm Biotechnol,
2004. 5(2): pp. 199-204. FCS and FCCS measurements provide two important
analytical parameters, the average number of molecules in the detection area
and the translational diffusion constant of the molecules at the single
molecule level. Considering these properties, FCS and FCCS have been applied to
analysis of the cellular environment and dynamic processes of molecules in the
living cell. More recently, a systematic approach for the analysis of
macromolecule complex formation has focused on the new field of single molecule
detection in the post-genome era. In this work, we tested the sensitivities of
FCS and FCCS based on the distance between fluorophores in DNA as a model
macromolecule complex. The results show that FCCS is not limited by the size of
the macromolecular complex even in a very small detection area.
Antibodies in
diagnostic applications. Tetin, S.Y. and Stroupe, S.D., Curr Pharm Biotechnol, 2004. 5(1):
pp. 9-16. Immunoassays, or assays that are using antibodies as the specific
binding reagents, have become one of the most common methodologies in
diagnostic laboratories. In this paper we review different configurations of
immunoassays as applied to a variety of analytes and sensitivity limits, along
with common detection techniques and strategies. Progress in developing of
ultra high affinity antibodies as a direction to improved immunoassays is also
reviewed. Finally, we specifically concentrate on determination of antibody
binding constants and performing immunoassays at the single molecule level using
Fluorescence Correlation Spectroscopy (FCS). This technique has become a
powerful tool in molecular binding characterizations and assay development, and
possibly will grow into a quantitative analytical method suitable for
diagnostic tests.
Monitoring human
parvovirus B19 virus-like particles and antibody complexes in solution by
fluorescence correlation spectroscopy. Toivola, J., Michel, P.O., Gilbert, L., Lahtinen, T.,
Marjomaki, V., Hedman, K., Vuento, M., and Oker-Blom, C., Biol Chem,
2004. 385(1): pp. 87-93. Fluorescence correlation spectroscopy (FCS) was
used in monitoring human parvovirus B19 virus-like particle (VLP) antibody
complexes from acute phase and past-immunity serum samples. The Oregon Green
488-labeled VLPs gave an average diffusion coefficient of 1.7 x 10(-7) cm2
s(-1) with an apparent hydrodynamic radius of 14 nm. After incubation of the
fluorescent VLPs with an acute phase serum sample, the mobility information
obtained from the fluorescence intensity fluctuation by autocorrelation analysis
showed an average diffusion coefficient of 1.5 x 10(-8) cm2 s(-1),
corresponding to an average radius of 157 nm. In contrast, incubation of the
fluorescent VLPs with a past-immunity serum sample gave an average diffusion
coefficient of 3.5 x 10(-8) cm2 s(-1) and a radius of 69 nm. A control serum
devoid of B19 antibodies caused a change in the diffusion coefficient from 1.7
x 10(-7) to 1.6 x 10(-7) cm2 s(-1), which is much smaller than that observed
with acute phase or past-immunity sera. Thus, VLP-antibody complexes with
different diffusion coefficients could be identified for the acute phase and
past-immunity sera. FCS measurement of VLP-immune complexes could be useful in
distinguishing between antibodies present in acute phase or past-immunity sera
as well as in titration of the VLPs.
Protein folding by
the effects of macromolecular crowding. Tokuriki, N., Kinjo, M., Negi, S., Hoshino, M., Goto, Y.,
Urabe, I., and Yomo, T., Protein Sci, 2004. 13(1): pp. 125-33.
Unfolded states of ribonuclease A were used to investigate the effects of
macromolecular crowding on macromolecular compactness and protein folding. The
extent of protein folding and compactness were measured by circular dichroism
spectroscopy, fluorescence correlation spectroscopy, and NMR spectroscopy in
the presence of polyethylene glycol (PEG) or Ficoll as the crowding agent. The
unfolded state of RNase A in a 2.4 M urea solution at pH 3.0 became native in
conformation and compactness by the addition of 35% PEG 20000 or Ficoll 70. In
addition, the effects of macromolecular crowding on inert macromolecule
compactness were investigated by fluorescence correlation spectroscopy using
Fluorescence-labeled PEG as a test macromolecule. The size of
Fluorescence-labeled PEG decreased remarkably with an increase in the
concentration of PEG 20000 or Ficoll 70. These results show that macromolecules
are favored compact conformations in the presence of a high concentration of
macromolecules and indicate the importance of a crowded environment for the folding
and stabilization of globular proteins. Furthermore, the magnitude of the
effects on macromolecular crowding by the different sizes of background
molecules was investigated. RNase A and Fluorescence-labeled PEG did not become
compact, and had folded conformation by the addition of PEG 200. The effect of
the chemical potential on the compaction of a test molecule in relation to the
relative sizes of the test and background molecules is also discussed.
Single-nucleotide
polymorphism detection using nanomolar nucleotides and single-molecule
fluorescence.
Twist, C.R., Winson, M.K., Rowland, J.J., and Kell, D.B., Anal Biochem,
2004. 327(1): pp. 35-44. We have exploited three methods for
discriminating single-nucleotide polymorphisms (SNPs) by detecting the incorporation
or otherwise of labeled dideoxy nucleotides at the end of a primer chain using
single-molecule fluorescence detection methods. Good discrimination of
incorporated vs free nucleotide may be obtained in a homogeneous assay (without
washing steps) via confocal fluorescence correlation spectroscopy or by
polarization anisotropy obtained from confocal fluorescence intensity
distribution analysis. Moreover, the ratio of the fluorescence intensities on
each polarization channel may be used directly to discriminate the nucleotides
incorporated. Each measurement took just a few seconds and was done in
microliter volumes with nanomolar concentrations of labeled nucleotides. Since
the confocal volumes interrogated are approximately 1fL and the reaction volume
could easily be lowered to nanoliters, the possibility of SNP analysis with
attomoles of reagents opens up a route to very rapid and inexpensive SNP
detection. The method was applied with success to the detections of SNPs that
are known to occur in the BRCA1 and CFTR genes.
Denaturation of dsDNA
by p53: fluorescence correlation spectroscopy study. Vukojevic, V., Yakovleva, T.,
Terenius, L., Pramanik, A., and Bakalkin, G., Biochem Biophys Res Commun,
2004. 316(4): pp. 1150-5. p53 activates transcription through
interaction with specific DNA sequences in gene promoters. It also regulates
DNA replication, recombination, and repair apparently through interactions with
DNA intermediates of these reactions. Biochemical activities relevant for these
functions of p53 include binding to the ends and internal segments of
single-stranded DNA molecules, catalysis of DNA renaturation, and strand
exchange. We report a novel activity of p53, its ability to denature double-stranded
DNA molecules aggregated by basic peptides. Stable complexes of coiled
single-stranded DNA molecules with basic peptides are formed in this reaction.
Thus, complementary to the ability to catalyze DNA renaturation, p53 denatures
double-stranded DNA when the latter reaction is thermodynamically favorable.
This p53 activity, along with its ability to interact physically with DNA
helicases, may be relevant for resolving double-stranded DNA intermediates and
inhibition of DNA recombination, which is critical for guarding of the genome.
Fluorescence
correlation spectroscopy investigation of a GFP mutant-enhanced cyan
fluorescent protein and its tubulin fusion in living cells with two-photon
excitation. Wang,
Z., Shah, J.V., Chen, Z., Sun, C.H., and Berns, M.W., J Biomed Opt,
2004. 9(2): pp. 395-403. This study investigates the feasibility of
using the enhanced cyan mutant of green fluorescent protein (ECFP) as a probe
for two-photon fluorescence correlation spectroscopy (FCS). Molecular dynamics
and other properties of ECFP and an ECFP-tubulin fusion protein were
investigated in living Potorous tridactylis (PTK2) cells. ECFP has high
molecular brightness in the nucleus (eta=3.3 kcpsm) and in the cytoplasm (3.2
kcpsm) under our experimental conditions. The diffusion constants of ECFP were
determined to be 20+/-7 microm(2)/s in the nucleus and 21+/-8 microm(2)/s in
the cytoplasm. ECFP has stable molecular characteristics with negligible
photobleaching and photodynamic effects in our measurements. At the highest concentration
of monomer ECFP (425 nM) the amount of dimer ECFP was estimated to be
negligible ( approximately 1.8 nM), consistent with our data analysis using a
single species model. ECFP-tubulin has a diffusion constant of 6 microm(2)/s in
the living cells. In addition, we demonstrate that analysis of the molecular
brightness can provide a new avenue for studying the polymerization state of
tubulin. We suggest that the tubulin in the vicinity of the nucleus exists
primarily as a heterodimer subunit while those in the area away from the
nucleus (d>5 microm) are mostly oligomers. We conclude that ECFP is a useful
genetic fluorescent probe for FCS studies of various cellular processes when in
fusion to other biomolecules of interest.
Molecular dynamics of
STAT3 on IL-6 signaling pathway in living cells. Watanabe, K., Saito, K., Kinjo,
M., Matsuda, T., Tamura, M., Kon, S., Miyazaki, T., and Uede, T., Biochem
Biophys Res Commun, 2004. 324(4): pp. 1264-73. Signal transducer and
activator of transcription 3 (STAT3) is a critical signal transducer of
interleukin-6 (IL-6) signaling. To investigate the mobility and the dynamics of
STAT3 complex on IL-6 signaling in living cells, we generated a chimeric gene
consisting of STAT3 fused to enhanced green fluorescence protein, STAT3-GFP.
STAT3-GFP was expressed in Hep3B cells and the dynamics of this protein were
analyzed by fluorescence correlation spectroscopy. After IL-6 stimulation,
STAT3 translocated from the cytoplasm to the nucleus, as shown previously.
According to the analysis of STAT3 diffusion in stable transformants, the
number of STAT3 molecules at the cytoplasmic membrane and in the cytoplasm
decreased after IL-6 stimulation. In the nucleus, the diffusion speed of STAT3
complex strongly decreased after IL-6 stimulation. Furthermore, we found that
STAT3 existed as a complex whose molecular weight was less than 400kDa before
IL-6 addition. However, IL-6 stimulation induced the formation of STAT3 dimer
as a megacomplex form whose molecular weight was more than 1MDa at the
cytoplasm and a very slow diffusion complex in the nucleus.
Anomalous
subdiffusion is a measure for cytoplasmic crowding in living cells. Weiss, M., Elsner, M., Kartberg,
F., and Nilsson, T., Biophys J, 2004. 87(5): pp. 3518-24.
Macromolecular crowding dramatically affects cellular processes such as protein
folding and assembly, regulation of metabolic pathways, and condensation of
DNA. Despite increased attention, we still lack a definition for how crowded a
heterogeneous environment is at the molecular scale and how this manifests in
basic physical phenomena like diffusion. Here, we show by means of fluorescence
correlation spectroscopy and computer simulations that crowding manifests
itself through the emergence of anomalous subdiffusion of cytoplasmic
macromolecules. In other words, the mean square displacement of a protein will
grow less than linear in time and the degree of this anomality depends on the
size and conformation of the traced particle and on the total protein
concentration of the solution. We therefore propose that the anomality of the
diffusion can be used as a quantifiable measure for the crowdedness of the
cytoplasm at the molecular scale.
The LSM 510 META -
ConfoCor 2 system: an integrated imaging and spectroscopic platform for single-molecule
detection.
Weisshart, K., Jungel, V., and Briddon, S.J., Curr Pharm Biotechnol,
2004. 5(2): pp. 135-54. Fluorescence Correlation Spectroscopy (FCS) has
developed into a routine method to quantitatively study diffusion of molecules
and kinetic processes. FCS has recently become popular to complement live cell
imaging with biophysical information. The enabling technology has been
commercially realised by combining laser scanning microscopes and fluorescence
correlation spectrometers in one integrated platform. This article provides an
overview of the Zeiss solution of such a combined instrument, the LSM 510 META
/ ConfoCor 2 system. We focus on the instrumental set up as well as technical
advances and improvements in the software that controls FCS data acquisition
and evaluation. In addition, we outline the calibration of the instrument and
the work flow for data analysis with emphasis on in vivo pharmacological
applications.
Physicochemical
characterisation of cationic polybutylcyanoacrylate-nanoparticles by
fluorescence correlation spectroscopy. Weyermann, J., Lochmann, D., Georgens, C., Rais, I.,
Kreuter, J., Karas, M., Wolkenhauer, M., and Zimmer, A., Eur J Pharm
Biopharm, 2004. 58(1): pp. 25-35. The aim of this study was to
compare different physical and chemical methods with fluorescence correlation
spectroscopy (FCS) in order to characterise cationic acrylate nanoparticles
(NP), which can deliver oligonucleotides (ON) into mammalian cells. These
positively charged nanoparticles were prepared from diethylaminoethyl dextran
(DEAE-dextran) and poly(n-butyl-2-cyanoacrylate) (PBCA). NP consists of PBCA
oligochains with an average size of PBCA 9 mer and were formed by entrapping
DEAE-dextran and dextran 70,000 in high amounts into the particle matrix. The
oligochain length of PBCA was investigated by mass-spectroscopy (MALDI TOF).
The molecular weight of a particle with d = 108 nm was estimated to be
approximately 3.6 x 10(8) Da. The mean size of the nanoparticles were in a
range of dh = 130-140 nm, as determined independently by FCS and dynamic light
scattering. Atomic force microscopy and scanning electron microscopy images
confirm this size range. Furthermore, the particle mass of the PBCA-NP was
estimated by FCS measurements. For this approach two new methods for
fluorescence labelling of cationic particles were developed. Fluorescent
labelled dextran 70,000 was entrapped into the particle matrix; in addition,
the derivatisation of hydroxyl groups of the NP was achieved with
5-([4,6-dichlorotriazin-2-yl]amino) fluorescein (DTAF). ON can be localised in
a complex with the NP by dual-colour fluorescence cross correlation
spectroscopy measurements. The zetapotential of the unloaded NP was positively
charged with about +39 mV and decreased down to -40 mV on addition of excess
ON. After centrifugation quantification of the ON loading onto the particles by
strong anion exchange high performance liquid chromatography (SAX HPLC) and FCS
showed that approximately 20 microg ON per 100 g NP was adsorbed. The FCS
measurements of the ON adsorption in situ was found to be much higher with
approximately 95 microg ON per 100 g NP.
Self-diffusion in
solutions of a 20 base pair oligonucleotide: effects of concentration and ionic
strength. Wilk, A.,
Gapinski, J., Patkowski, A., and Pecora, R., J Chem Phys, 2004. 121(21):
pp. 10794-802. The long-time self-diffusion coefficients of a 20 base pair
duplex oligonucleotide are measured as functions of 20-mer and added NaCl salt
concentrations. The self-diffusion coefficients decrease monotonically with
increasing 20-mer concentrations for the high-added salt sample and display
non-monotonically decreasing 20-mer concentration dependences at lower added
salt concentrations. The non-monotonic behavior is attributed to the opposing
effects of the tendency to increase the interactions between 20-mers as the
concentration is increased and to a decrease in the extent of the Coulomb
forces as counterions from the 20-mer increasingly screen them. Attempts to
account for the effect of the Coulomb forces on the self-diffusion coefficients
by using effective dimensions in the hard rod theory give good agreement with
experiment at the highest salt concentration studied. For the lower salt
concentrations there appear to be two scaling regimes--one at low polyion
concentration in which the high salt scaling of the rod dimensions by adding
the Debye screening to the length and diameter of the rod is appropriate and
one at high polyion concentrations where the scaling of the dimensions is the addition
of 1/2 the Debye screening length. Estimates of the "overlap"
concentration C*=1/L(eff) indicate that the non-monotonic decrease occurs at
concentrations lower than C*. Finally, the fluorescence correlation
spectroscopy self-diffusion coefficients measured here are compared with the
mutual diffusion coefficients measured by dynamic light scattering.
Direct gene
expression analysis.
Winter, H., Korn, K., and Rigler, R., Curr Pharm Biotechnol, 2004. 5(2):
pp. 191-7. The direct analysis of single biological molecules is getting
increasingly important in basic as well as pharmaceutical research (e.g. for
gene expression analysis). In particular single-molecule fluorescence detection
provides exciting new opportunities to probe biochemical processes in unprecedented
detail. Currently several academic and industrial research groups work on the
development of single molecule detection based technologies in order to
directly detect and analyze RNA and DNA molecules. As these developed methods
are characterized as homogenous assays and obviate any amplification of the
target or the signal, they provide clear advantages compared to methods like
real-time PCR or DNA- arrays. In the following we describe a recently developed
approach based on fluorescence correlation spectroscopy (FCS). This expression
assay is based on gene-specific hybridization of two dye-labeled DNA probes to
a selected target molecule (either DNA or RNA) in solution. The subsequent dual
color cross-correlation analysis allows the quantification of the bio-molecule
of interest in absolute numbers. Target concentrations of less than 10(-12) M
can be easily monitored, covering the direct analysis of the expression levels
of high, medium and low abundant genes.
Endothelin receptor
in virus-like particles: ligand binding observed by fluorescence fluctuation
spectroscopy.
Zemanova, L., Schenk, A., Hunt, N., Nienhaus, G.U., and Heilker, R., Biochemistry,
2004. 43(28): pp. 9021-8. The functional analysis of transmembrane
receptor proteins is frequently hampered by the difficulty to produce
sufficiently homogeneous receptor preparations that preserve the physiological
biomembrane integration of the receptor protein. To improve the receptor
protein density in the lipid bilayer and to maintain the physiological
lipid-protein environment, a novel method has been established that enables the
selective integration of transmembrane receptors into a virus-like particle
(VLiP). Here we have studied the binding of tetramethylrhodamine-labeled
endothelin-1 (TMR-ET-1) to VLiP-integrated endothelin A receptor (ET(A)R) by
fluorescence fluctuation spectroscopy. The concentration of TMR-ET-1 was
determined by fluorescence correlation spectroscopy (FCS). These measurements
also confirmed that the free ligand is monomeric in solution in our
experiments. Fluorescence intensity distribution analysis (FIDA) was used to
quantify the fraction of ligands bound to ET(A)Rs in the VLiPs. For the
interaction between ET-1 and VLiP-integrated ET(A)Rs, K(D) values of 0.5 nM and
0.3 nM were determined from ligand and receptor titration experiments,
respectively. For comparison, a FIDA analysis was also carried out with ET(A)Rs
in membrane fragments derived from an ET(A)R-overexpressing mammalian cell
line, which yielded a similar K(D) of 0.2 nM. In addition, we examined the
binding competition of a set of reference compounds to VLiP-ET(A)Rs in the
presence of ET-1 and obtained K(i) values similar to those reported in the
literature. Our results demonstrate that integration into VLiPs does not change
the binding properties of the ET(A)Rs. FIDA analysis of VLiP-integrated
receptors shows great promise for highly miniaturized and fast compound testing
in the pharmaceutical industry.
Fluorescence microscopy studies with
a fluorescent glibenclamide derivative, a high-affinity blocker of pancreatic
beta-cell ATP-sensitive K+ currents. Zunkler, B.J., Wos-Maganga, M., and Panten, U., Biochem
Pharmacol, 2004. 67(8): pp. 1437-44. Hypoglycemic sulfonylureas (e.g.
tolbutamide, glibenclamide) exert their stimulatory effects on pancreatic
beta-cells by closure of ATP-sensitive K(+) (K(ATP)) channels. Pancreatic
K(ATP) channels are composed of two subunits, a pore-forming inwardly
rectifying K(+) channel (Kir6.2) subunit and a regulatory subunit (the
sulfonylurea receptor of subtype 1 (SUR1)) in a (SUR1/Kir6.2)(4) stoichiometry.
The aim of the present study was to characterize the interaction of
green-fluorescent 3-[3-(4,4 difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-S-indacen-3-yl)propanamido]
glibenclamide (Bodipy-glibenclamide) with pancreatic beta-cell K(ATP) channels
using patch-clamp and fluorescence microscopy techniques. Bodipy-glibenclamide
inhibited K(ATP) currents from the clonal insulinoma cell line RINm5F half-maximally
at a concentration of 0.6nM. Using laser-scanning confocal microscopy
Bodipy-glibenclamide was shown to induce a diffuse fluorescence across the
RINm5F cell, but only about 17% of total Bodipy-glibenclamide-induced
fluorescence intensity in RINm5F cells was due to specific binding to SUR1.
Using fluorescence correlation spectroscopy, it could be demonstrated that the
fluorescence label contributes to the protein binding and, therefore, possibly
also to the non-specific binding of Bodipy-glibenclamide observed in RINm5F
cells. Specific binding of Bodipy-glibenclamide to SUR1 in RINm5F cells might
be localized to different intracellular structures (nuclear envelope,
endoplasmic reticulum, Golgi compartment, insulin secretory granules) as well as
to the plasma membrane. In conclusion, Bodipy-glibenclamide is a high-affinity
blocker of pancreatic beta-cell K(ATP) currents and can be used for visualizing
SUR1 in intact pancreatic beta-cells, although non-specific binding must be
taken into account in confocal microscopy experiments on intact beta-cells.
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2004 FCS abstracts |