Thermodynamics and kinetics of protein-protein interactions for proteins involved in protein ubiquitination

The figure on the right shows a superposition of ¹H-¹⁵N NMR spectra of the protein Mms2 upon successive additions of the protein ubiquitin. The backbone amide cross peaks whose chemical shifts change give an the approximate location of the ubiquitin binding site on Mms2. In addition, we can plot the chemical shift changes as a function of the ligand:protein ratio to determine a dissociation constant.

New NMR methods for thermodynamics and kinetics of protein-ligand interactions

NMR is an ideal method for determining the dissociation constants K_D of weak protein-ligand interactions. For a typical NMR titration, the initial protein concentration is held nearly constant, and for this condition, the protein concentration needs to be about half the dissociation constant to determine an accurate K_D value, and the binding site needs to be saturated with ligand at the end of the titration, this requires a lot of ligand. We developed methods where co-variation of the protein and ligand concentrations gives more precise K_D values, and accurate and precise kinetics.