Fluorescence Correlation Spectroscopy Measurements Research Articles

Characterization of the Surface Contribution to Fluorescence Correlation Spectroscopy Measurements

Fluorescence correlation spectroscopy (FCS) is a sophisticated and an accurate analytical technique used to study the diffusion of molecules in a solution at the single-molecule level. FCS is strongly affected by many factors such as the stability of the excitation power, photochemical processes, mismatch between the refractive indices, and variations in the cover glass thickness. We have studied FCS near the surface of a cover glass by using rhodamine 123 as a fluorescent probe and have observed that the surface has a strong influence on the measurements. The temporal autocorrelation of FCS decays with two characteristic times when the confocal detection volume is positioned near the surface of the cover glass. As the position of the detection volume is moved away from the surface, the FCS autocorrelation becomes one-component decaying; the characteristic time of the decay is the same as the faster-decaying component in the FCS autocorrelation near the surface. This observation suggests that the faster component can be attributed to the free diffusion of the probe molecules in the solution, while the slow component has its origin from the interaction between the probe molecules and the surface. We have characterized the surface contribution to the FCS measurements near the surface by changing the position of the detection volume relative to the surface. The influence of the surface on the diffusion of the probe molecules was monitored by changing the chemical properties of the surface. The surface contribution to the temporal autocorrelation of the FCS strongly depends on the chemical nature of the surface. The hydrophobicity of the surface is a major factor determining the surface influence on the free diffusion of the probe molecules near the surface.

Fluorescence Correlation Spectroscopy and Dynamic Light Scattering Measurements in Ficoll Solutions

We apply fluorescence correlation spectroscopy (FCS) and dynamic light scattering (DLS) to measure both the translational diffusion coefficient, D, of different fluorescent probes embedded in Ficoll solutions and the density fluctuations of the Ficoll solutions. Ficoll (MW=70 kDa) is a water-soluble, branched polysaccharide usually considered to be a spherical nanoparticle. The probes (R6G, dextran10, R-Erythrin proteins, clathrin, polystyrene beads,..) vary not only in size (1.8 nm to 34 nm) but also in shape (globular, branched polymers, triskelia). We find, using FCS, that the decrease of D as a function of Ficoll concentration (up to 500 mg/ml) can be described by an exponential, exp(-αc), with α being dependent on the probe size. This can interpreted within a model proposed by DeGennes and co-workers, which predicts a stretched exponential, D ∼exp(-αc-ν), with ν being related to the solvent quality and α proportional to the probe size. Thus, ν≈1, indicating a θsolvent like quality for the water-Ficoll system. The DLS measurements were performed as a function of the scattering angle and Ficoll concentration. At low concentrations (< 1mg/ml), we determined the hydrodynamic diameter of the Ficoll to be about 11 nm. As the Ficoll concentration is increased, a second mode appears in the correlation function, indicating possible interactions between the Ficoll polymers. We fitted the correlations with double stretched exponentials and determined the relaxation times of both modes as a function of the concentration. While one mode increases by several orders of magnitude, the other decreases weakly and exponentially, whose prefactor depends on the scattering angle. Estimates based on packing criteria of spherical beads show that the Ficoll polymers must intertwine in order to fit the volumetric space, indicating that Ficoll polymers are not compact nanoparticles and confirming the observations from DLS.

Objective, Bayesian Analysis of Fluorescence Correlation Spectroscopy Data

Fluctuations in fluorescence intensity provide quantitative insight into a range of molecular processes including diffusion, active transport, and binding kinetics in living cells and tissues. Classical fluorescence correlation spectroscopy (FCS) measures fluctuations in fluorescence intensity in a small detection volume to infer molecular transport properties from governing continuum diffusion-convection equations. Essential to the interpretation of FCS data is the use of an underlying mathematical model that governs the molecular process under study. Autocorrelations in fluctuations in fluorescence intensity may then be fit to the analytical solution of the model, and corresponding molecular properties may be inferred. While the choice of physical process may be unambiguous for special cases of free molecular diffusion or convective transport in solution, the correct choice of model becomes considerably less unambiguous in the application of FCS to complex biological processes in living cells or extracellular matrices. For this reason, an objective and unbiased approach to model selection and parameter estimation is of interest. The method of Bayesian inference provides such a framework. At the level of parameter estimation, Bayesian inference is similar to Maximum Likelihood Estimation when flat priors are chosen for initial parameter estimates. At the level of model selection, however, Bayesian inference assigns explicit model probabilities that are proportional to their marginal likelihoods by considering the full range of parameter values and their posterior probability distributions rather than only using point-wise, Maximum Likelihood estimates, thereby appropriately penalizing model complexity and preventing over-fitting of experimental data. Here we illustrate application of Bayesian inference to the problem of model selection and parameter estimation of molecular transport properties from FCS data, comparing it to traditional model selection approaches.

Fluorescence Correlation Spectroscopy of Fast Chain Dynamics within Denatured Protein L

Intramolecular dynamics in the denatured state of a protein are of importance for protein folding. Native-like contact formation within the ensemble of denatured conformations of a protein may guide its transformation towards the native conformation. The efficiency of folding is thus dependent on the diffusion of chain fragments, which facilitates contact formation. Herein we investigate intramolecular diffusion of denatured molecules of the small two-state-folding protein L with fluorescence correlation spectroscopy (FCS). We utilize the specific quenching of the fluorescence of the oxazine dye Atto655 labeling a cysteine at position 64 (the C-terminus of the protein) by the side chain of a tryptophan at position 47. FCS measurements allow us to probe processes ranging in timescales from tens of nanoseconds to seconds. Two fast photophysical processes can be distinguished in the fluorescence correlation curves. The slower of the two is found to be due to triplet dynamics, while the faster process is attributed to the quenching of the Atto655 by the tryptophan upon transient ground-state complex formation. We study the dependence of the intrachain dynamics of the denatured protein on the concentration of the denaturant guanidinium chloride (GdmCl), and extract complex association and dissociation rates. While the dissociation rate does not depend on the denaturant, the association rate decreases as denaturant concentration is increased from 3 to 7 M GdmCl. This decrease in contact formation rate tracks the expansion of denatured protein L, measured in our previous work. Thus, the intramolecular diffusion coefficient calculated from the results is found to be essentially independent of the denaturant concentration over this range, even as the protein expands by more than 20%.

On the performance of bioanalytical fluorescence correlation spectroscopy measurements in a multiparameter photon-counting microscope

Fluorescence correlation spectroscopy (FCS) data acquisition and analysis routines were developed and implemented in a home-built, multiparameter photon-counting microscope. Laser excitation conditions were investigated for two representative fluorescent probes, Rhodamine110 and enhanced green fluorescent protein (EGFP). Reliable local concentrations and diffusion constants were obtained by fitting measured FCS curves, provided that the excitation intensity did not exceed 20% of the saturation level for each fluorophore. Accurate results were obtained from FCS measurements for sample concentrations varying from pM to μM range, as well as for conditions of high background signals. These experimental constraints were found to be determined by characteristics of the detection system and by the saturation behavior of the fluorescent probes. These factors actually limit the average number of photons that can be collected from a single fluorophore passing through the detection volume. The versatility of our setup and the data analysis capabilities were tested by measuring the mobility of EGFP in the nucleus of Drosophila cells under conditions of high concentration and molecular crowding. As a bioanalytical application, we studied by FCS the binding affinity of a novel peptide-based drug to the cancer-regulating STAT3 protein and corroborated the results with fluorescence polarization analysis derived from the same photon data.

Novel Multiparametric Approach to Elucidate the Surface Amine-Silanization Reaction Profile on Fluorescent Silica Nanoparticles

This Article addresses the important issue of the characterization of surface functional groups for optical bioassay applications. We use a model system consisting of spherical dye-doped silica nanoparticles (NPs) that have been functionalized with amine groups whereby the encapsulated cyanine-based near-infrared dye fluorescence acts as a probe of the NP surface environment. This facilitates the identification of the optimum deposition parameters for the formation of a stable ordered amine monolayer and also elucidates the functionalization profile of the amine-silanization process. Specifically, we use a novel approach where the techniques of fluorescence correlation spectroscopy (FCS) and fluorescence lifetime measurement (FL) are used in conjunction with the more conventional analytical techniques of zeta potential measurement and Fourier transfer infrared spectroscopy (FTIR). The dynamics of the ordering of the amine layer in different stages of the reaction have been characterized by FTIR, FL, and FCS. The results indicate an optimum reaction time for the formation of a stable amine layer, which is optimized for further biomolecular conjugation, whereas extended reaction times lead to a disordered cross-linked layer. The results have been validated using an immunoglobulin (IgG) plate-based direct binding assay where the maximum number of IgG-conjugated aminated NPs were captured by immobilized anti-IgG antibodies for the NP sample corresponding to the optimized amine-silanization condition. Importantly, these results point to the potential of FCS and FL as useful analytical tools in diverse fields such as characterization of surface functionalization.

Interplay between Structure and Fluidity of Model Lipid Membranes under Oxidative Attack

A proper regulation of membrane fluidity is critical for cellular activities such as communication between cells, mitosis, and endocytosis. Unsaturated lipids, a main component of biological membranes, are particularly susceptible to oxidative attack of reactive oxygen species. The oxidation of lipids can produce structural derangement of membranes and eventually alter the membrane fluidity. We have applied fluorescence correlation spectroscopy (FCS) and Raman spectroscopy to investigate the fluidity and structure of model membranes subject to oxidative attack. Hydrogen peroxide has little effect on the lateral fluidity of membranes, whereas hydroxyl radical causes a significantly increased fluidity. The latter is rationalized with the cleavage of the acyl chains of lipids caused by hydroxyl radical; this interpretation is founded on the diminished intensities of lines in Raman spectra associated with -CH(2) and C═C moieties in lipids and supported by mass-spectral measurements. The same approach provides a mechanistic account of the inhibitory capability of vitamins C and E against the increased membrane fluidity resulting from an oxidative attack. Membranes with much cholesterol exhibit a novel resistance against altered membrane fluidity induced with oxidative attack; this finding has biological implications. Our approach combining FCS and Raman measurements reveals the interplay between the structure and fluidity of membranes and provides insight into the pathophysiology of cellular oxidative injury.

Cross-Validating FRAP and FCS to Quantify the Impact of Photobleaching on In Vivo Binding Estimates

Binding can now be quantified in live cells, but the accuracy of such measurements remains uncertain. To address this uncertainty, we compare fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) measurements of the binding kinetics of a transcription factor, the glucocorticoid receptor, in the nuclei of live cells. We find that the binding residence time measured by FRAP is 15 times longer than that obtained by FCS. We show that this discrepancy is not likely due to the significant differences in concentrations typically used for FRAP and FCS, nor is it likely due to spatial heterogeneity of the nucleus, improper calibration of the FCS focal volume, or the intentional FRAP photobleach. Instead, our data indicate that photobleaching of bound molecules in FCS is mainly responsible. When this effect is minimized, FRAP and FCS measurements nearly agree, although cross-validation by other approaches is now required to rule out mutual errors. Our results demonstrate the necessity of a photobleach correction for FCS measurements of GFP-tagged molecules that are bound for >0.25 s, and represent an important step forward in establishing a gold standard for in vivo binding measurements.

A Closed Form for Fluorescence Correlation Spectroscopy Experiments in Submicrometer Structures

Fluorescence correlation spectroscopy (FCS) is a powerful technique for measuring low concentrations of fluorescent molecules and their diffusion coefficients in an open detection volume. However, in several practical cases, when FCS measurements are carried out in small compartments like microchannels, neglecting boundary effects could lead to erroneous results. Here, a close form solution is proposed to explicitly account for the presence of walls located at a distance comparable with the characteristic detection volume lengths. We derive a one-dimensional diffusion constrained model and then generalize the solution to the two- and the three-dimensional constrained cases. We further indicate within which limits the standard autocorrelation function (ACF) model gives reliable results in microconfinement. Our model relies just on the assumption of elastic hits at the system walls and succeeds in describing the ACF of fluorescent probes confined along one direction. Through the analysis of FCS experimental data, we are able to predict the correct shape of the ACF in channels of micrometric and submicrometric width and measure the extent of lateral confinement. In addition, it permits the investigation of microstructured material features such as cages and cavities having dimensions on the micrometric range. On the basis of the proposed model, we also show in which conditions confinement could generate an apparent time dependent probe mobility, thus allowing a proper interpretation of the transport process taking place in submicrometric compartments.

Identification of Protein-Protein and Protein-Ribosome Interacting Regions of the C-terminal Tail of Human Mitochondrial Inner Membrane Protein Oxa1L

The mammalian mitochondrial inner membrane protein Oxa1L is involved in the insertion of a number of mitochondrial translation products into the inner membrane. During this process, the C-terminal tail of Oxa1L (Oxa1L-CTT) binds mitochondrial ribosomes and is believed to coordinate the synthesis and membrane insertion of the nascent chains into the membrane. The C-terminal tail of Oxa1L does not contain any Cys residues. Four variants of this protein with a specifically placed Cys residue at position 4, 39, 67, or 94 of Oxa1L-CTT have been prepared. These Cys residues have been derivatized with a fluorescent probe, tetramethylrhodamine-5-maleimide, for biophysical studies. Oxa1L-CTT forms oligomers cooperatively with a binding constant in the submicromolar range. Fluorescence anisotropy and fluorescence lifetime measurements indicate that contacts near a long helix close to position 39 of Oxa1L-CTT occur during oligomer formation. Fluorescence correlation spectroscopy measurements demonstrate that all of the Oxa1L-CTT derivatives bind to mammalian mitochondrial ribosomes. Steady-state fluorescence quenching and fluorescence lifetime data indicate that there are extensive contacts between Oxa1L-CTT and the ribosome-encompassing regions around positions 39, 67, and 94. The results of this study suggest that Oxa1L-CTT undergoes conformational changes and induced oligomer formation when it binds to the ribosome.

Measuring and imaging diffusion with multiple scan speed image correlation spectroscopy

The intracellular mobility of biomolecules is determined by transport and diffusion as well as molecular interactions and is crucial for many processes in living cells. Methods of fluorescence microscopy like confocal laser scanning microscopy (CLSM) can be used to characterize the intracellular distribution of fluorescently labeled biomolecules. Fluorescence correlation spectroscopy (FCS) is used to describe diffusion, transport and photo-physical processes quantitatively. As an alternative to FCS, spatially resolved measurements of mobilities can be implemented using a CLSM by utilizing the spatio-temporal information inscribed into the image by the scan process, referred to as raster image correlation spectroscopy (RICS). Here we present and discuss an extended approach, multiple scan speed image correlation spectroscopy (msICS), which benefits from the advantages of RICS, i.e. the use of widely available instrumentation and the extraction of spatially resolved mobility information, without the need of a priori knowledge of diffusion properties. In addition, msICS covers a broad dynamic range, generates correlation data comparable to FCS measurements, and allows to derive two-dimensional maps of diffusion coefficients. We show the applicability of msICS to fluorophores in solution and to free EGFP in living cells.

Slow molecular dynamics close to crystal surfaces during crystallization of a protein lysozyme studied by fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) was applied to the crystallization processes of egg-white lysozyme. Utilizing FCS's high spatial resolution of about the laser wavelength used, the molecular dynamics close to crystal surfaces was investigated for both tetragonal single crystals and needlelike spherulites. When the FCS measurement was done at the point closer than 1 μm to the surface of a tetragonal single crystal, the relaxation time became several times longer than that in bulk solution, but the fluorescence intensity (thus concentration) was similar to that observed in bulk solution. On the other hand, the peculiar slow dynamics (a few orders of magnitude slower than that in bulk solution) of concentrated liquid states of the lysozyme molecules was observed in needlelike spherulites. We suggested that these observations could be explained by the formation of softly connected aggregates accumulating around the needlelike crystals, which could cause the instability of the crystal growth and thus the formation of spherulites. These aggregates gradually disappeared as the crystallization further proceeded. After the disappearance of the aggregates, the spherulites started to mature.

Iodide as a Fluorescence Quencher and Promoter—Mechanisms and Possible Implications

In this work, fluorescence correlation spectroscopy (FCS) was used to investigate the effects of potassium iodide (KI) on the electronic-state population kinetics of a range of organic dyes in the visible wavelength range. Apart from a heavy atom effect promoting intersystem crossing to the triplet states in all dyes, KI was also found to enhance the triplet-state decay rate by a charge-coupled deactivation. This deactivation was only found for dyes with excitation maximum in the blue range, not for those with excitation maxima at wavelengths in the green range or longer. Consequently, under excitation conditions sufficient for triplet state formation, KI can promote the triplet state buildup of one dye and reduce it for another, red-shifted dye. This anticorrelated, spectrally separable response of two different dyes to the presence of one and the same agent may provide a useful readout for biomolecular interaction and microenvironmental monitoring studies. In contrast to the typical notion of KI as a fluorescence quencher, the FCS measurements also revealed that when added in micromolar concentrations KI can act as an antioxidant, promoting the recovery of photo-oxidized fluorophores. However, in millimolar concentrations KI also reduces intact, fluorescently viable fluorophores to a considerable extent. In aqueous solutions, for the dye Rhodamine Green, an optimal concentration of KI of approximately 5 mM can be defined at which the fluorescence signal is maximized. This concentration is not high enough to allow full triplet state quenching. Therefore, as a fluorescence enhancement agent, it is primarily the antioxidative properties of KI that play a role.

Diffusion Measurements inside Biofilms by Image-Based Fluorescence Recovery after Photobleaching (FRAP) Analysis with a Commercial Confocal Laser Scanning Microscope

Research about the reactional and structural dynamics of biofilms at the molecular level has made great strides, owing to efficient fluorescence imaging methods in terms of spatial resolution and fast acquisition time but also to noninvasive conditions of observation consistent with in situ biofilm studies. In addition to conventional fluorescence intensity imaging, the fluorescence recovery after photobleaching (FRAP) module can now be routinely implemented on commercial confocal laser scanning microscopes (CLSMs). This method allows measuring of local diffusion coefficients in biofilms and could become an alternative to fluorescence correlation spectroscopy (FCS). We present here an image-based FRAP protocol to improve the accuracy of FRAP measurements inside "live" biofilms and the corresponding analysis. An original kymogram representation allows control of the absence of perturbing bacterial movement during image acquisition. FRAP data analysis takes into account molecular diffusion during the bleach phase and uses the image information to extract molecular diffusion coefficients. The fluorescence spatial intensity profile analysis used here for the first time with biofilms is supported both by our own mathematical model and by a previously published one. This approach was validated to FRAP experiments on fluorescent-dextran diffusion inside Lactococcus lactis and Stenotrophomonas maltophilia biofilms, and the results were compared to previously published FCS measurements.

Evaluation of radiation force acting on macromolecules by combination of Brownian dynamics simulation with fluorescence correlation spectroscopy

The effect of optical gradient force from a focused laser beam on the fluorescence correlation spectroscopy (FCS) was investigated by a computing method based on Brownian dynamics simulation. A series of calculations revealed that, in relatively shallow optical force potential up to 1.0kTR (TR=298.15 K), the conventional theoretical model of FCS without consideration of the optical gradient force could evaluate the increase in the average number of molecules and the diffusion time in the potential. On the other hand, large deviation between the simulated fluorescence correlation curve and the theoretical model was observed under the potential depth >1.0kTR. In addition, by integrating the optical force potential with the temperature elevation under optical trapping condition, it was deduced that the temperature rise does not seriously affect the average number of particles in the sampling area, but the average residence time is more sensitively affected by the temperature elevation. The present study using the simulation also provides a method to experimentally estimate molecular polarizabilities from FCS measurements.

Thermosensitive hydrogel-containing polymersomes for controlled drug delivery

PNIPAAm-containing polymersomes (N/Ps) were prepared by injecting a solution of poly(ethylene glycol)-b-poly(D,L-lactide) (mPEG-PDLLA) and poly(N-isopropylacrylamide) (PNIPAAm) in THF into water to incorporate PNIPAAm into polymersomes (Ps). At 37 degrees C, hydrogel-containing Ps (Hs, hydrosomes) with an average diameter of 127 nm as measured with dynamic light scattering (DLS) were obtained which may be used as potential novel carriers for anticancer drugs and proteins. Dual-labeled N/Ps (FITC-N/RB-Ps) were prepared analogously using rhodamine B tagged mPEG-PDLLA (mPEG-PDLLA-RB) and fluorescein isothiocyanate labeled PNIPAAm (FITC-N). The co-localization of RB labeled Ps (RB-Ps) and FITC-N in RB-Ps was shown by dual fluorescence CLSM. Fluorescence correlation spectroscopy (FCS) and fluorescence anisotropy (FA) measurements with these systems gave further evidence for the colocalization of PNIPAAm and Ps. Micron-sized giant Ps with a diameter of 5-10 microm containing FITC-N were prepared using CHCl(3) as the organic phase. The presence of FITC-N in these giant Ps as well as the phase separation of the internal FITC-N solution above the lower critical solution temperature (LCST) was also shown by CLSM. The release of fluorescein isothiocyanate tagged dextran (FD, FITC-dextran, Mw 4000 g/mol) from Hs revealed that in the presence of the hydrogel at 37 degrees C a more sustained release of FD (up to 30 days) with a low initial burst effect was obtained as compared to the release from bare Ps.

Characterization of Horizontal Lipid Bilayers as a Model System to Study Lipid Phase Separation

Artificial lipid membranes are widely used as a model system to study single ion channel activity using electrophysiological techniques. In this study, we characterize the properties of the artificial bilayer system with respect to its dynamics of lipid phase separation using single-molecule fluorescence fluctuation and electrophysiological techniques. We determined the rotational motions of fluorescently labeled lipids on the nanosecond timescale using confocal time-resolved anisotropy to probe the microscopic viscosity of the membrane. Simultaneously, long-range mobility was investigated by the lateral diffusion of the lipids using fluorescence correlation spectroscopy. Depending on the solvent used for membrane preparation, lateral diffusion coefficients in the range Dlat = 10–25 μm2/s and rotational diffusion coefficients ranging from Drot = 2.8 − 1.4 × 107 s−1 were measured in pure liquid-disordered (Ld) membranes. In ternary mixtures containing saturated and unsaturated phospholipids and cholesterol, liquid-ordered (Lo) domains segregated from the Ld phase at 23°C. The lateral mobility of lipids in Lo domains was around eightfold lower compared to those in the Ld phase, whereas the rotational mobility decreased by a factor of 1.5. Burst-integrated steady-state anisotropy histograms, as well as anisotropy imaging, were used to visualize the rotational mobility of lipid probes in phase-separated bilayers. These experiments and fluorescence correlation spectroscopy measurements at different focal diameters indicated a heterogeneous microenvironment in the Lo phase. Finally, we demonstrate the potential of the optoelectro setup to study the influence of lipid domains on the electrophysiological properties of ion channels. We found that the electrophysiological activity of gramicidin A (gA), a well-characterized ion-channel-forming peptide, was related to lipid-domain partitioning. During liquid-liquid phase separation, gA was largely excluded from Lo domains. Simultaneously, the number of electrically active gA dimers increased due to the increased surface density of gA in the Ld phase.

Automated suppression of sample-related artifacts in Fluorescence Correlation Spectroscopy

Fluorescence Correlation Spectroscopy (FCS) in cells often suffers from artifacts caused by bright aggregates or vesicles, depletion of fluorophores or bleaching of a fluorescent background. The common practice of manually discarding distorted curves is time consuming and subjective. Here we demonstrate the feasibility of automated FCS data analysis with efficient rejection of corrupted parts of the signal. As test systems we use a solution of fluorescent molecules, contaminated with bright fluorescent beads, as well as cells expressing a fluorescent protein (ICA512-EGFP), which partitions into bright secretory granules. This approach improves the accuracy of FCS measurements in biological samples, extends its applicability to especially challenging systems and greatly simplifies and accelerates the data analysis.

Detecting Intramolecular Dynamics and Multiple Förster Resonance Energy Transfer States by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy (FCS) is a robust method for the detection of intramolecular dynamics in proteins but is also susceptible to interference from other dynamic processes such as triplet kinetics and photobleaching. We describe an approach for the detection of intramolecular dynamics in proteins labeled with a FRET dye pair based on global fitting to the two autocorrelation functions (green-green and red-red) and the two cross-correlation functions (green-red and red-green). We applied the method to detect intramolecular dynamics in the Ca(2+) signaling protein calmodulin. Dynamics were detected on the 100 mus time scale in Ca(2+)-activated calmodulin, whereas in apocalmodulin dynamics were not detected on this time scale. Control measurements on a polyproline FRET construct (Gly-Pro(15)-Cys) demonstrate the reliability of the method for isolating intramolecular dynamics from other dynamic processes on the microsecond time scale and confirm the absence of intramolecular dynamics of polyproline. We further show the sensitivity of the initial amplitudes of the FCS auto- and cross-correlation functions to the presence of multiple FRET states, static or dynamic. The FCS measurements also show that the diffusion of Ca(2+)-calmodulin is slower than that of apocalmodulin, indicating either a larger average hydrodynamic radius or shape effects resulting in a slower translational diffusion.

The effect of the sn‐2 acyl chain of diacylglycerol on its interaction with C1 domains and its signaling role

Diacylglycerol (DAG) is a signaling lipid that interacts with the C1 domain in various target proteins, including protein kinases C, and Ras guanyl nucleotide‐releasing proteins, thereby recruiting them to the plasma membrane. Although these proteins can all bind DAG, some are known to target other intracellular membranes. To understand the basis of this differential membrane targeting, we explored the possibility that a difference in the acyl chain composition of DAG in the plasma membrane and other membranes may affect the C1‐DAG interaction. To test this notion, we measured the binding of the various C1 domains to DAGs with different sn‐2 acyl groups by fluorescence correlation spectroscopy (FCS). The C1 domains were expressed as an enhanced green fluorescence protein‐fusion protein to enhance protein stability and allow FCS measurements. FCS measurements as well as the molecular dynamics simulation show that the sn‐2 acyl group of DAG has significant and differential effects on the membrane binding properties of C1 domains, which is consistent with the differential subcellular localization of the C1 domains.Supported by NIH GM76581