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

Characterization of Fluorescently Labeled Protein with Electrospray Ionization-MS and Fluorescence Spectroscopy: How Random is Random Labeling?

Solvent exposed lysine residues are abundantly present in many proteins. Their highly reactive ε-amino groups serve as universal targets for coupling with active esters of various extrinsic labels including a vast arsenal of fluorescent probes. Here, we describe fluorescent properties and preferential localization of two frequently used fluorescent labels, AlexaFluor488 (AF488) and Cy3, on the surface of a small highly soluble serum protein neutrophil gelatinase-associated lipocalin (NGAL), which serves as a diagnostic marker for acute kidney failure. Using a standard protocol for labeling with either AF488-SDP or AF488-NHS, we achieved >95% labeling efficiency of the protein as determined by UV–vis absorption and electrospray ionization (ESI)-MS. However, fluorescence intensity of the labeled protein was less than 10% of the expected value. To understand the unusually high quenching of the probe, we identified the sites of AF488 attachments by means of LC-MS/MS combined with trypsin digestion. Surprisingly, we found that the AF488 label is not randomly distributed among accessible lysines but predominantly bound to the residues K125, K126, or K135, which are located in the NGAL calyx and are likely quenched by neighboring tryptophans and tyrosines. In contrast, when NGAL was labeled with Cy3, the probe’s fluorescence was almost fully retained. The LC-MS/MS data indicated that Cy3 was predominately bound to another lysine, K31, on the protein surface on the opposite side of the calyx. Our findings suggest that a combination of the inherent properties of the label and the specifics of the protein microenvironment may selectively lead probes to specific lysine residues and thus challenge the common view that protein labeling is a random process

Introduction of the Mass Spread Function for Characterization of Protein Conjugates

Traditionally, characterization of protein molecules conjugated with molecular probes is performed by UV–vis spectroscopy. This method determines the average incorporation ratio but does not yield information about the label distribution. Electrospray ionization mass spectroscopy (ESI-MS) allows direct measurement of the fraction of protein containing a given number of labels. However, for a glycosylated protein, this analysis can be severely limited due to spectral overlap of the labels and carbohydrates. To address this problem, we introduce the mass spread function (MSF) for conjugation analysis. By treating the ESI-MS spectrum of conjugated protein as the spectrum before conjugation convolved with the MSF, we are able to quantify the labeled protein population using a binomial distribution function. We first applied this procedure for characterization of labeled antibody F­(ab′)₂ fragments which do not contain carbohydrates. We then apply the MSF to fit spectra of entire conjugated monoclonal antibodies and quantify the distribution of labels in the presence of glycans