Time-Integrated Fluorescence Cumulant Analysis (TIFCA) Enables Model Selection in Ligand Binding Studies

Abstract

TIFCA is a fluorescence fluctuation spectroscopy technique that makes use of both the molecular brightness and diffusion time of freely diffusing fluorescent species. TIFCA was introduced to calculate statistically significant higher order cumulants to accurately describe protein interactions using rigorous fitting procedures. Previously, we have applied TIFCA to perform a titration, confirm antibody stoichiometry and measure the binding affinity by first assuming a bivalent antibody model. The cumulants were calculated up to forth order and globally fit to resolve the fraction of bound and unbound ligand. The global fitting routine uses all points in the titration simultaneously and returns a global reduced chi2 for the entire titration series. It was found that analysis of the global reduced chi2 alone is not sensitive to the chosen binding model. Here we show that TIFCA has the ability to determine proper binding models by evaluating fits to individual cumulants within the titration series. Using a small peptide and antibody, we show that local reduced chi2 values are rather high for expressions describing a monovalent antibody. Only the correct number of species describing the bivalent nature of antibodies results in acceptable local reduced chi2 values for all orders of cumulants. More specifically, it is found that the second order cumulant is highly dependent upon the number of binding sites in an antibody molecule leading to the correct binding model.J.D.M. acknowledges support from the National Institute of Health (GM64589).