Paternalism or partnership?

<h3>Abstract</h3> 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 <i>trp</i>RNA binding attenuation protein, TRAP. First, we developed nearest-neighbor statistical thermodynamic binding models comprising microscopic free energies for ligand binding to isolated sites ΔG_<b>N0</b>, and for coupling between one or both adjacent sites, ΔG_N1 and ΔG_N2. Using the resulting partition function (PF) we explored the effects of these parameters on simulated population distributions for the 2^N possible liganded states. We then experimentally monitored liganddependent population shifts using conventional spectroscopic and calorimetric methods, and using native mass spectrometry (MS). By resolving species with differing numbers of bound ligands by their mass, native MS 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. Uniquely, this approach predicts <i>which</i> of the possible 2^N liganded states are populated at different ligand concentrations, providing necessary insights into regulation. The combination of statistical thermodynamic modeling with native MS may provide the thermodynamic foundation for a meaningful understanding of the structure-thermodynamic linkage that drives cooperativity. <h3>TOC Figure (draft)</h3>