Sergey Y. Tetin

Probing Ligand Protein Binding Equilibria with Fluorescence Fluctuation Spectroscopy

We examine the binding of fluorescent ligands to proteins by analyzing the fluctuation amplitude g(0) of fluorescence fluctuation experiments. The normalized variance g(0) depends on the molecular brightness and the concentration of each species in the sample. Thus a single g(0) measurement is not sufficient to resolve individual species. Titration of the ligand with protein establishes the link between molecular brightness and concentration by fitting g(0) to a binding model and allows the separation of species. We first apply g(0) analysis to binary dye mixtures with brightness ratios of 2 and 4 to demonstrate the feasibility of this technique. Next we consider the influence of binding on the fluctuation amplitude g(0). The dissociation coefficient, the molecular brightness ratio, and the stochiometry of binding strongly influence the fluctuation amplitude. We show that proteins with a single binding site can be clearly differentiated from proteins with two independent binding sites. The binding of fluorescein-labeled digoxigenin to a high-affinity anti-digoxin antibody was studied experimentally. A global analysis of the fluctuation amplitude and the fluorescence intensity not only recovered the dissociation coefficient and the number of binding sites, but also revealed the molecular heterogeneity of the hapten-antibody complex. Two species were used to model the molecular heterogeneity. We confirmed the molecular heterogeneity independently by fluorescence lifetime experiments, which gave fractional populations and molecular brightness values that were virtually identical to those of the g(0) analysis. The identification and characterization of molecular heterogeneity have far-reaching consequences for many biomolecular systems. We point out the important role fluctuation experiments may have in this area of research.

Accuracy of protein secondary structure determination from circular dichroism spectra based on immunoglobulin examples

Strong contribution of the aromatic amino acid side chain chromophores to the far-UV circular dichroism (CD) spectra substantially distorts a relatively weak CD signal originating from beta sheet, the main type of immunoglobulin secondary structure. In this study we compared the secondary structure calculated from the far-UV CD spectra with the X-ray data for three antibody Fab fragments. Calculations were performed with three different algorithms, using two sets of reference proteins. Low standard deviations between all six estimates indicate stable mathematical solutions. Despite pronounced differences in the shape and amplitude of the CD spectra, we found a strong correlation between CD and X-ray data in the secondary structure for every protein studied. The number and average length of the secondary structure elements estimated from the CD spectra closely resemble those of the X-ray data. Agreement between spectroscopic and crystallographic results demonstrates that modern methods of secondary structure calculation are resilient to distortions of the far-UV CD spectra of immunoglobulins caused by aromatic side chain chromophores.

Measuring antibody affinity and performing immunoassay at the single molecule level

Fluorescence correlation spectroscopy (FCS) enables direct observation of the translational diffusion of single fluorescent molecules in solution. When fluorescent hapten binds to antibody, analysis of FCS data yields the fractional amounts of free and bound hapten, allowing determination of the equilibrium binding constant. Equilibrium dissociation constants of anti-digoxin antibodies and corresponding fluorescein-labeled digoxigenin obtained by FCS and fluorescence polarization measurements are identical. It is also possible to follow a competitive displacement of the tracer from the antibody by unlabeled hapten using FCS in an immunoassay format. The fluorescence polarization immunoassay for vancomycin detection was used to test the FCS approach. Fitting of the FCS data for the molar fractions of free and bound fluorescein-labeled vancomycin yielded a calibration curve which could serve for determination of the vancomycin concentration in biological samples.

Using nonfluorescent Förster resonance energy transfer acceptors in protein binding studies.

The purpose of this article is to highlight the versatility of nonfluorescent Förster resonance energy transfer (FRET) acceptors in determination of protein equilibrium dissociation constants and kinetic rates. Using a nonfluorescent acceptor eliminates the necessity to spectrally isolate the donor fluorescence when performing binding titrations covering a broad range of reagent concentrations. Moreover, random distribution of the donor and acceptor chromophores on the surface of proteins increases the probability of FRET occurring on their interaction. Three high-affinity antibodies are presented in this study as characteristic protein systems. Monoclonal antibody (mAb) 106.3 binds brain natriuretic peptide (BNP)5-13(C10A) and full-length BNP1-32 with the dissociation constants 0.26+/-0.01 and 0.05+/-0.02 nM, respectively, which was confirmed by kinetic measurements. For anti-hCG (human chorionic gonadotropin) mAb 8F11, studied at two incorporation ratios (IRs=1.9 and 3.8) of the nonfluorescent FRET acceptor, K(D) values of 0.04+/-0.02 and 0.059(-0.004)(+0.006) nM, respectively, were obtained. Likewise, the binding of goat anti-hamster immunoglobulin G (IgG) antibody was not affected by conjugation and yielded K(D) values of 1.26+/-0.04, 1.25+/-0.05, and 1.14+/-0.04 nM at IRs of 1.7, 4.7, and 8.1, respectively. We conclude that this FRET-based method offers high sensitivity, practical simplicity, and versatility in protein binding studies.

Simplified confocal microscope for counting particles at low concentrations

We describe a compact scanning confocal fluorescence microscope capable of detecting particles concentrations less than 100 particles∕ml in ~15 min. The system mechanically moves a cuvette containing ~3 ml of sample. A relatively large confocal volume is observed within the cuvette using a 1 mm pinhole in front of a detection PMT. Due to the motion of the sample, particles traverse the confocal volume quickly, and analysis by pattern recognition qualifies spikes in the emission intensity data and counts them as events. We show linearity of detection as a function of concentration and also characterize statistical behavior of the instrument. We calculate a detection sensitivity of the system using 3 μm fluorescent microspheres to be 5 particles/ml. Furthermore, to demonstrate biological application, we performed a dilution series to quantify stained E. coli and yeast cells. We counted E. coli cells at a concentration as low as 30 cells∕ml in 10 min/sample.

Epitope Mapping of Anti-DCP Antibody Using Fluorescence Correlation Spectroscopy

Des-gamma-carboxy prothrombin (DCP), also known as protein induced by vitamin K antagonist (PIVKA-II), is a recognized clinical marker for hepatocellular carcinoma. Prothrombin contains 10 gluamic acid (Glu) residues within its N-terminus (GLA domain) which are post modified to gamma-carboxyglutamic acid (GLA). DCP is an abnormal form of prothrombin in which some of the 10 glutamic acid residues remain unmodified. A monoclonal antibody was developed to specifically recognize DCP, but not prothrombin. In this study we identified the epitope of the anti-DCP antibody using a series of short peptides representing the GLA domain. For each peptide, a single Glu residue was replaced with a GLA residue. The critical Glu residues recognized by the antibody were identified in a competitive format using fluorescence correlation spectroscopy (FCS). We also evaluated the DCP specificity of the antibody using homologues peptide from various GLA domain proteins present in various blood coagulation factors. The dissociation constant of the antibody was determined using FRET based method.

Random Fluorescently Labeled Proteins: Label Distribution and Effect on Binding

Proteins for fluorescence measurements are often labeled randomly by covalent linkage of fluorescent dyes to amine groups on the target protein and subsequently purified. Such labeling results in a heterogeneous population of protein molecules containing a varied number of labels, which may depend on the number and location of available lysine residues. We explore the extent to which protein labeling techniques result in a Poissonian distribution of protein-fluorophore complexes using fluorescence fluctuation spectroscopy (FFS). The fluctuation amplitude in an FFS measurement is related to the number of labeled proteins and is not sensitive to unlabeled protein. We model the expected fluctuation amplitude as a function of average incorporated fluorophores assuming the distribution is governed by Poissonian statistics. We experimentally fit the model by randomly labeling monoclonal antibody with fluorescent dye and show agreement for incorporation ratios up to ∼ 1.5. For greater amounts of incorporated dye molecules, we use mass spectrometry to examine labeled F(ab’)2 fragments and show that the distribution is better described by a Gaussian profile. Finally, by performing quenching experiments on a steady-state fluorimeter, we show that randomly labeling antibodies and antigens does not affect measured affinity values within experimental uncertainty.

Strategy And Biophysical Tools For Developing Modern Diagnostic Assays

Neutrophil Gelatinase-Associated Lipocalin (NGAL) is a 20 kDa monomeric protein secreted by activated human neutrophils. NGAL is believed to bind small lipophilic substances such as bacteria-derived lipopolysaccharides, siderophores, formylpeptides, and may function as a modulator of inflammation. Clinical studies have shown NGAL can serve as an early diagnostic marker for acute kidney injury (AKI). We have developed a sensitive immunoassay to measure NGAL level in patient urine. During the course of assay development, we employed a variety of biophysical methods to characterize the physical and binding properties of several anti-NGAL antibody candidates and a recombinant NGAL protein. CD spectroscopy was used to study the structure and stability of NGAL; Forster Resonance Energy Transfer (FRET) was used to determine the binding affinity of NGAL toward anti-NGAL mAbs; Dual-Color Fluorescence Cross-Correlation Spectroscopy (DC-FCCS) was used to compare the capability of two antibodies forming a sandwich with NGAL; NMR was used to identify epitopic regions of NGAL for each antibody candidate. Two antibodies, which have the highest binding affinity toward NGAL and recognize distinct discontinuous epitope regions on NGAL, were chosen as the reagents for a sandwich type microparticle based immunoassay. This work demonstrates a modern strategy and biophysical tools, which are necessary to build a sensitive and robust diagnostic assay.

Kinetics and Thermodynamics of Antibody Binding to B-Type Natriuretic Peptide

B-type natriuretic peptide (BNP) is a naturally secreted regulatory hormone that influences blood pressure and vascular water retention. The plasma BNP concentration is a clinically recognized biomarker for various cardiovascular diseases. Quantitative detection of BNP can be achieved in immunoassays using high-affinity monoclonal antibodies. Temperature dependence of the equilibrium binding constants and the kinetic rates were studied for anti-BNP mAbs 106.3 and 3-631 by means of fluorescence spectroscopy. Thermodynamic parameters including changes in the free energy, enthalpy and entropy measured at equilibrium are in a good agreement with the parameters calculated from kinetics data. The differences in thermodynamic parameters measured for the two antibodies under study support structural data obtained by NMR and X-ray crystallography.