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.
Chen
Y
Y
Laboratory for Fluorescence Dynamics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA.
Müller
J D
JD
Tetin
S Y
SY
Tyner
J D
JD
Gratton
E
E
D000911:Antibodies, Monoclonal / Q000737:chemistry*; D001665:Binding Sites; D001666:Binding Sites, Antibody; D004077:Digoxin / Q000276:immunology; D005456:Fluorescent Dyes; D006241:Haptens / Q000737:chemistry*; D007700:Kinetics; D008024:Ligands; D011506:Proteins / Q000737:chemistry*; D013050:Spectrometry, Fluorescence / Q000379:methods
D000911:Antibodies, Monoclonal; D005456:Fluorescent Dyes; D006241:Haptens; D008024:Ligands; D011506:Proteins; D004077:Digoxin