Fluorescence correlation spectroscopy study of protein transport and dynamic interactions with clustered-charge peptide adsorbents

Ion-exchange chromatography relies on electrostatic interactions between the adsorbent and the adsorbate and is used extensively in protein purification. Conventional ion-exchange chromatography uses ligands that are singly charged and randomly dispersed over the adsorbent, creating a heterogeneous distribution of potential adsorption sites. Clustered-charge ion exchangers exhibit higher affinity, capacity, and selectivity than their dispersed-charge counterparts of the same total charge density. In the present work, we monitored the transport behavior of an anionic protein near clustered-charge adsorbent surfaces using fluorescence correlation spectroscopy. We can resolve protein-free diffusion, hindered diffusion, and association with bare glass, agarose-coated, and agarose-clustered peptide surfaces, demonstrating that this method can be used to understand and ultimately optimize clustered-charge adsorbent and other surface interactions at the molecular scale.     

Fluorescence correlation spectroscopy in membrane structure elucidation

This review describes the application of fluorescence correlation spectroscopy (FCS) for the study of biological membranes. Monitoring the fluorescence signal fluctuations, it is possible to obtain diffusion constants and concentrations for several membrane components. Focusing the attention on lipid bilayers, we explain the technical difficulties and the new FCS-based methodologies introduced to overcome them. Finally, we report several examples of studies which apply FCS on both model and biological membranes to obtain interesting insight in the topic of lateral membrane organization.     

Fluorescence correlation spectroscopy as a new method for the investigation of aptamer/target interactions

Fluorescence correlation spectroscopy is an attractive tool for monitoring molecular interactions in solution. We report here a new and highly sensitive method for studying the interaction of aptamers with their targets using this technique. In vitro selection technology is a combinatorial method for the generation of nucleic acid receptors (aptamers) that are capable of binding to various target molecules. Using the in vitro selection approach we isolated RNAs which bind to the antibiotic moenomycin with high affinity. The formation of RNA-moenomycin complexes was studied by fluorescence correlation spectroscopy with a tetramethylrhodamine-labeled derivative of moenomycin.     

Fluorescence correlation spectroscopy analysis of the hydrophobic interactions of protein 4.1 with phosphatidyl serine liposomes

Fluorescence correlation spectroscopy (FCS) was applied to examine the interactions between a protein and a membrane lipid. The protein 4.1-phosphatidyl serine (PS) interactions served as the model system to demonstrate the membrane lipid-protein interactions. This protein was labeled with rhodamine and its interactions with PS-liposomes were measured by FCS. The present results clearly demonstrated that a small protein molecule, protein 4.1, interacts specifically with a large particle, a PS-liposome. This interaction appears to be hydrophobic and not electrostatic, since the bound protein 4.1 did not dissociate in solution and was specifically released from PS-liposomes by treatment with phospholipase A(2) (PLA(2)). In the present study, using FCS we could demonstrate that the serine residue of PS is required for protein 4.1 to bind to PS-liposomes and that the bound protein 4.1 is closely associated with the fatty acid of the PS molecule in the liposomes.     

CGDB: A database of membrane protein/lipid interactions by coarse-grained molecular dynamics simulations

Membrane protein function and stability has been shown to be dependent on the lipid environment. Recently, we developed a high-throughput computational approach for the prediction of membrane protein/lipid interactions. In the current study, we enhanced this approach with the addition of a new measure of the distortion caused by membrane proteins on a lipid bilayer. This is illustrated by considering the effect of lipid tail length and headgroup charge on the distortion caused by the integral membrane proteins MscS and FLAP, and by the voltage sensing domain from the channel KvAP. Changing the chain length of lipids alters the extent but not the pattern of distortion caused by MscS and FLAP; lipid headgroups distort in order to interact with very similar but not identical regions in these proteins for all bilayer widths investigated. Introducing anionic lipids into a DPPC bilayer containing the KvAP voltage sensor does not affect the extent of bilayer distortion.     

CGDB: a database of membrane protein/lipid interactions by coarse-grained molecular dynamics simulations

Membrane protein function and stability has been shown to be dependent on the lipid environment. Recently, we developed a high-throughput computational approach for the prediction of membrane protein/lipid interactions. In the current study, we enhanced this approach with the addition of a new measure of the distortion caused by membrane proteins on a lipid bilayer. This is illustrated by considering the effect of lipid tail length and headgroup charge on the distortion caused by the integral membrane proteins MscS and FLAP, and by the voltage sensing domain from the channel KvAP. Changing the chain length of lipids alters the extent but not the pattern of distortion caused by MscS and FLAP; lipid headgroups distort in order to interact with very similar but not identical regions in these proteins for all bilayer widths investigated. Introducing anionic lipids into a DPPC bilayer containing the KvAP voltage sensor does not affect the extent of bilayer distortion.     

Fluorescence correlation spectroscopy close to a fluctuating membrane

Compartmentalization of the cytoplasm by membranes should have a strong influence on the diffusion of macromolecules inside a cell, and we have studied how this could be reflected in fluorescence correlation spectroscopy (FCS) experiments. We derived the autocorrelation function measured by FCS for fluorescent particles diffusing close to a soft membrane, and show it to be the sum of two contributions: short timescale correlations come from the diffusion of the particles (differing from free diffusion because of the presence of an obstacle), whereas long timescale correlations arise from fluctuations of the membrane itself (which create intensity fluctuations by modulating the number of detected particles). In the case of thermal fluctuations this second type of correlation depends on the elasticity of the membrane. To illustrate this calculation, we report the results of FCS experiments carried out close to a vesicle membrane. The measured autocorrelation functions display very distinctly the two expected contributions, and allow both to recover the diffusion coefficient of the fluorophore and to characterize the membrane fluctuations in term of a bending rigidity. Our results show that FCS measurements inside cells can lead to erroneous values of the diffusion coefficient if the influence of membranes is not recognized.     

Fluorescence techniques to study lipid dynamics

Biological research has always tremendously benefited from the development of key methodology. In fact, it was the advent of microscopy that shaped our understanding of cells as the fundamental units of life. Microscopic techniques are still central to the elucidation of biological units and processes, but equally important are methods that allow access to the dimension of time, to investigate the dynamics of molecular functions and interactions. Here, fluorescence spectroscopy with its sensitivity to access the single-molecule level, and its large temporal resolution, has been opening up fully new perspectives for cell biology. Here we summarize the key fluorescent techniques used to study cellular dynamics, with the focus on lipid and membrane systems.     

Characterizing diffusion dynamics of a membrane protein associated with nanolipoproteins using fluorescence correlation spectroscopy

Nanolipoprotein particles (NLPs) represent a unique nanometer-sized scaffold for supporting membrane proteins (MP). Characterization of their dynamic shape and association with MP in solution remains a challenge. Here, we present a rapid method of analysis by fluorescence correlation spectroscopy (FCS) to characterize bacteriorhodopsin (bR), a membrane protein capable of forming a NLP complex. By selectively labeling individual components of NLPs during cell-free synthesis, FCS enabled us to measure specific NLP diffusion times and infer size information for different NLP species. The resulting bR-loaded NLPs were shown to be dynamically discoidal in solution with a mean diameter of 7.8 nm. The insertion rate of bR in the complex was ～55% based on a fit model incorporating two separate diffusion properties to best approximate the FCS data. More importantly, based on these data, we infer that membrane protein associated NLPs are thermodynamically constrained as discs in solution, while empty NLPs appear to be less constrained and dynamically spherical.     

Fluorescence correlation spectroscopy for the detection and study of single molecules in biology

The recent development of single molecule detection techniques has opened new horizons for the study of individual macromolecules under physiological conditions. Conformational subpopulations, internal dynamics and activity of single biomolecules, parameters that have so far been hidden in large ensemble averages, are now being unveiled. Herein, we review a particular attractive solution-based single molecule technique, fluorescence correlation spectroscopy (FCS). This time-averaging fluctuation analysis which is usually performed in Confocal setups combines maximum sensitivity with high statistical confidence. FCS has proven to be a very versatile and powerful tool for detection and temporal investigation of biomolecules at ultralow concentrations on surfaces, in solution, and in living cells. The introduction of dual-color cross-correlation and two-photon excitation in FCS experiments is currently increasing the number of promising applications of FCS to biological research.     

Fluorescence correlation spectroscopy analysis of the dynamics of tubulin interaction with RB3, a stathmin family protein

We have used fluorescence correlation spectroscopy to analyze the interaction of GTP-tubulin with rhodamine-labeled RB3, a neural protein of the stathmin family, and to determine the kinetic pathway of the association process. RB3 displayed slow association-dissociation kinetics with tubulin depending on the square of the tubulin concentration. The values of the apparent association and dissociation rate constants of the complex of two tubulin dimers and RB3 are determined to be (3.52+/-0.14)x10(-3) micro;M(-2)/s and (1.9+/-0.6)x10(-3) s(-1) respectively. The value of the equilibrium dissociation constant for the first tubulin-RB3 interaction is estimated to be >or=7 microM at 20 degrees C.     

Fluorescence correlation spectroscopy diffusion laws to probe the submicron cell membrane organization

To probe the complexity of the cell membrane organization and dynamics, it is important to obtain simple physical observables from experiments on live cells. Here we show that fluorescence correlation spectroscopy (FCS) measurements at different spatial scales enable distinguishing between different submicron confinement models. By plotting the diffusion time versus the transverse area of the confocal volume, we introduce the so-called FCS diffusion law, which is the key concept throughout this article. First, we report experimental FCS diffusion laws for two membrane constituents, which are respectively a putative raft marker and a cytoskeleton-hindered transmembrane protein. We find that these two constituents exhibit very distinct behaviors. To understand these results, we propose different models, which account for the diffusion of molecules either in a membrane comprising isolated microdomains or in a meshwork. By simulating FCS experiments for these two types of organization, we obtain FCS diffusion laws in agreement with our experimental observations. We also demonstrate that simple observables derived from these FCS diffusion laws are strongly related to confinement parameters such as the partition of molecules in microdomains and the average confinement time of molecules in a microdomain or a single mesh of a meshwork.     

Fluorescence correlation spectroscopy can probe albumin dynamics inside lung endothelial glycocalyx

The endothelial glycocalyx is believed to play a major role in capillary permeability by functioning as a macromolecular barrier overlying the intercellular junction. Little is known about the functional attributes of the glycocalyx (i.e., porosity and permeability) or which constituents contribute to its overall structure-function relationship. In this report, we demonstrate the utility of fluorescence correlation spectroscopy (FCS) to measure albumin diffusion rates and concentration profiles above the cell surface and overlying the intercellular junctions of lung capillary endothelial cells. Albumin diffusion rates and concentration profiles were obtained before and after enzymatic digestion of the glycocalyx with pronase, heparanase, or hyaluronidase. The results suggest a structure interacting with albumin located from 1.0 to 2.0 microm above the cell membrane capable of reducing albumin diffusion by 30% while simultaneously increasing albumin concentration fivefold. Digestion of the glycocalyx with pronase or heparanase resulted in only modest changes in albumin diffusion and concentration profiles. Hyaluronidase digestion completely eliminated albumin-glycocalyx interactions. These data also suggest that hyaluronan is a major determinant for albumin interactions with the lung endothelial glycocalyx. Confocal images of heparan sulfate and hyaluronan confirm a cell-surface layer 2-3 mum in thickness, thus supporting FCS measurements. In summary, we report the first use of FCS to probe extracellular structures and further our understanding of the structure-function relationship of the lung microvascular endothelial glycocalyx.     

Fluorescence correlation spectroscopy and nonideal solutions

The information that may be obtained from a fluorescence correlation spectroscopic study of a nonideal solution is considered. If all of the macromolecules in a two-component solution are fluorescently labeled, the mutual diffusion coefficient will be measured. If only a few of the macromolecules in a solution are fluorescently labeled, the tracer diffusion coefficient will be obtained. Two nonideal systems that probably may usefully be studied with fluorescence correlation spectroscopy are proposed. The application of fluorescence correlation spectroscopy to studies of lateral diffusion in biological membranes is discussed; the form of the contribution to the fluorescence correlation spectrum of bulk motion within a membrane is noted.     

Fluorescence correlation spectroscopy studies of Peptide and protein binding to phospholipid vesicles

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.     

Fluorescence correlation spectroscopy reveals topological segregation of the two tumor necrosis factor membrane receptors

The proinflammatory cytokine tumor necrosis factor (TNF) binds two distinct plasma membrane receptors, TNFR1 and TNFR2. We have produced different receptor mutants fused with enhanced green fluorescent protein to study their membrane dynamics by fluorescence correlation spectroscopy (FCS). TNFR1 mutants show diffusion constants of approximately 1.2 × 10^− 9 cm²/s and a broad distribution of diffusion times, which is hardly affected by ligand binding. However, cholesterol depletion enhances their diffusion, suggesting a constitutive affinity to cholesterol rich membrane microdomains. In contrast, TNFR2 and mutants thereof diffuse rather fast (D? = 3.1 × 10^− 9 cm²/s) with a marked reduction after 30 min of TNF treatment (D? = 0.9 × 10^− 9 cm²/s). This reduction cannot be explained by the formation of higher ordered receptor clusters, since the fluorescence intensity of TNF treated receptors indicate the presence of a few receptor molecules per complex only. Together, these data point to a topological segregation of the two TNF receptors in different microcompartments of the plasma membrane independent of the cytoplasmic signaling domains of the receptors.     

Ligand-receptor interactions in the membrane of cultured cells monitored by fluorescence correlation spectroscopy

We investigated the specific binding of epidermal growth factor (EGF) to its membrane-bound receptors in cultured cells. The specificity of the binding was attested by the consistent displacement of bound rhodamine-labeled EGF (Rh-EGF) following addition of 1000-fold molar excess of unlabeled EGF. The binding specificity of EGF was further confirmed when vascular EGF was unable to displace Rh-EGF binding, demonstrating no cross-reaction. Evidence for the specific interactions was verified by an equilibrium saturation binding experiment. EGF binding to the cell membranes is saturated at nanomolar concentration. The Scatchard plots show a binding process with K(ass) of 1.5 x 10(9) M(-1). The dissociation kinetics follow a single exponential function characteristic for a relatively slow dissociation process with k(diss) = 2.9 x 10(-4) s(-1). The appearance of two binding complexes through the distribution of diffusion times may suggest that these are representatives of two different forms or subtypes of EGF receptors. This study is of pharmaceutical significance as it provides evidence that fluorescence correlation spectroscopy can be used as a rapid technique for studying ligand-receptor interactions in cell cultures. This is a step forward toward large-scale drug screening in cell cultures.     

Fluorescence correlation spectroscopy as a method for assessment of interactions between phage displaying antibodies and soluble antigen

Phage display is widely used for expression of combinatorial libraries, not least for protein engineering purposes. Precise selection at the single molecule level will provide an improved tool for generating proteins with complex and distinct properties from large molecular libraries. To establish such an improved selection system, we here report the detection of specific interactions between phage with displayed antibody fragments and fluorescently labeled soluble antigen based on Fluorescence Correlation Spectroscopy (FCS). Our novel strategy comprises the use of two separate fluorochromes for detection of the phage-antigen complex, either with labeled antiphage antibody or using a labeled antigen. As a model system, we studied a human monoclonal antibody to the hepatitis-C virus (HCV) envelope protein E2 and its cognate antigen (rE2 or rE1/E2). We could thus assess the specific interactions and determine the fraction of specific versus background phage (26% specific phage). Aggregation of these particular antigens made it difficult to reliably utilize the full potential of cross-correlation studies using the two labels simultaneously. However, with true monomeric proteins, this will certainly be possible, offering a great advantage in a safer and highly specific detection system.     

Fluorescence correlation spectroscopy and its potential for intracellular applications

Fluorescence correlation spectroscopy (FCS) is a time-averaging fluctuation analysis of small molecular ensembles, combining maximum sensitivity with high statistical confidence. Among a multitude of physical parameters that are, in principle, accessible by FCS, it most conveniently allows to determine local concentrations, mobility coefficients, and characteristic rate constants of fast-reversible and slow-irreversible reactions of fluorescently labeled biomolecules at very low (nanomolar) concentrations, under equilibrium conditions and without physical separation. Its presently most popular instrumentation by confocal-microscope setups allows for a spatial resolution of fractions of femtoliters for the measurement volumes, containing sparse or even single molecules at any time, and encourages the adaptation of the solution-based technique for cellular applications. The scope of this review is thus, to introduce the FCS technique in particular to the reader with biological background, searching for new methods for a precise quantification of physical parameters governing cellular mechanisms and dynamics, especially if high sensitivity and fast dynamic resolution are required. After a short theoretical introduction, examples are given for the so far most important experimental applications, with respect to their implementation in cellular systems. As an interesting alternative to the confocal instrumentation, two-photon excitation will be introduced, offering a number of important advantages especially in cellular systems with high-noise and low-signal levels.