EQUILIBRIUM AND PREEQUILIBRIUM FLUORESCENCE SPECTROSCOPIC STUDIES OF THE BINDING OF A SINGLE-IMMUNOGLOBULIN-BINDING DOMAIN DERIVED FROM PROTEIN-G TO THE FC FRAGMENT FROM HUMAN IGG(1)

A single-immunoglobulin-binding protein based upon the C2 domain of Pr otein G from Streptococcus has been shown to bind tightly to the Fc fr agment of IgG(1). The binding interaction results in a decrease in the fluorescence intensity from the sole Trp residue (Trp-48) in this dom ain. This spectral change has been used to monitor the binding interac tions between the two proteins using equilibrium and pre-equilibrium f luorescence spectroscopy. Comparison of the data from the two techniqu es suggests that a conformational change occurs after the initial form ation of the complex. Mutagenesis studies have shown that the Trp resi due is important for binding and that replacement by a Phe residue lea ds to a 300-fold decrease in the affinity for Fc(gamma 1). Determinati on of the rate constants k(on) and k(off) at different values of pH be tween 4.0 and 9.0 suggest that variations in K-d are mediated predomin antly by changes in k(on). Competition experiments between SpG(1) and a single-IgG-binding domain from Protein A from Staphylococcus aureus have been used to determine the affinity of the latter for Fc(gamma 1) .