Linked equilibria in protein-protein binding: Thermodynamic modeling of contributions to the binding of a serine protease inhibitor to a serine protease

When studying protein-protein interactions it is often found that the overall binding equilibrium includes additional linked equilibria. This linkage will contribute to the binding thermodynamics and must be taken into account to best understand structure/energetic relationships. This requires high resolution structural data, appropriate experimental design and data analysis, along with representative thermodynamic models. Here a combination of experimental and computational approaches is used to address these issues.; Discrepancies between enthalpies determined calorimetrically, $DHocal$, and using the van't Hoff method, $DHovH$, have been noted in the literature. These discrepancies are addressed through simple experimental systems and numerical simulation of more complex binding equilibria. It is demonstrated that with appropriate data and error analysis, $DHocal$ and $DHovH$, are equivalent regardless of the nature of the linked equilibria, which includes proton-linked binding, as long as all equilibria are allowed to re-equilibrate with changing temperature.; The effects of linked equilibria, including proton binding and changes in dynamics upon binding, were investigated using the serine protease inhibitor, turkey ovomucoid third domain (OMTKY3) and the serine protease, subtilisin Carlsberg, to better understand linked contributions to observed protein-protein binding thermodynamics. The crystal structure and intrinsic binding thermodynamics of the complex are presented. It is found that unlike previously determined energetics for OMTKY3 binding the serine protease porcine pancreatic elastase, the OMTKY3/subtilisin Carlsberg complex possess a significantly favorable enthalpy.; The use of structure-based thermodynamic calculations suggests that the binding of OMTKY3 is not well represented by a rigid-body binding model. Examination of the unbound ensemble suggests that OMTKY3, when binding subtilisin Carlsberg, undergoes a transition to a more enthalpically favorable state at the price of lost conformational freedom. To better understand this contribution a new, ensemble-based approach is presented to account for dynamic contributions to binding events.; The presented model provides a framework in which to understand the thermodynamic contributions of protein dynamics. Expanding upon and improving this model will be essential in understanding binding reactions that differ from rigid-body binding.