Thermodynamic coupling between neighboring binding sites in homo-oligomeric ligand sensing proteins from mass resolved ligand dependent population distributions

Abstract

Homo-oligomeric ligand-activated proteins are ubiquitous in biology. The functions of such molecules are commonly regulated by allosteric coupling between ligand binding sites. Understanding the basis for this regulation requires both quantifying the free energy ΔG transduced between sites, and the structural basis by which it is transduced. We consider allostery in three variants of the model ring-shaped homo-oligomeric tryptophan-binding protein TRAP. First, we develop nearest-neighbor statistical thermodynamic binding models comprising microscopic free energies for ligand binding to isolated sites ΔGN0, and for coupling between one or both adjacent sites, ΔGN1 and ΔGN2. Using the resulting partition function (PF) we explore the effects of these parameters on simulated population distributions for the 2N possible liganded states. We then experimentally monitored ligand-dependent population shifts using conventional spectroscopic and calorimetric methods, and using native mass spectrometry (nMS). By resolving species with differing numbers of bound ligands by their mass, nMS revealed striking differences in their ligand-dependent population shifts. Fitting the populations to a binding polynomial derived from the PF yielded coupling free energy terms corresponding to orders of magnitude differences in cooperativity. The combination of statistical thermodynamic modeling with native mass spectrometry may provide the thermodynamic foundation for meaningful structure-thermodynamic understanding of cooperativity.