Elucidating slow binding kinetics of a protein without observable bound resonances by longitudinal relaxation NMR spectroscopy.

We developed a new method to elucidate the binding kinetics k(on) and k(off), and the dissociation constant K(D) (=k(off)/k(on)), of protein-protein interactions without observable bound resonances of the protein of interest due to high molecular weight in a complex with a large target protein. In our method, k(on) and k(off) rates are calculated from the analysis of longitudinal relaxation rates of free resonances measured for multiple samples containing different concentration ratios of (15)N-labeled protein and substoichiometric amounts of the target protein. The method is applicable to interactions that cannot be analyzed by relaxation dispersion spectroscopy due to slow interactions on millisecond to second timescale and/or minimal conformational (chemical shift) change upon binding. We applied the method to binding of the B1 domain of protein G (GB1) to immunoglobulin G, and derived the binding kinetics despite the absence of observable bound GB1 resonances.