Role of Conformational Entropy in Extremely High Affinity Protein Interactions

Abstract

Interactions of extreme affinity (Kd ∼ fM) underlie many biochemical processes necessary to life. The physical determinants of such large binding energies are not well understood. Specific interactions at the interface (ΔHbinding) and the release of solvating water (TΔSsolvation) are usually assumed to dominate the binding energetics. The role of conformational entropy (TΔSconf) in determining binding affinity has remained elusive, in part due to the difficulties in measuring such changes in entropy experimentally. Recent developments in the Wand laboratory have bridged this gap by using solution NMR measurements of dynamics to empirically calibrate a “conformational entropy meter.” It has enabled quantitative measurements of the change in conformational entropy in protein-ligand binding. The toxin-antitoxin system studied here, barnase-barstar, forms a complex with fM affinity (ΔGbinding ∼ 19 kcal/mol) without undergoing any major structural changes and retaining a hydrated interface. To explore the role of conformational entropy, the fast (ps-ns time-scale) motions of backbone and side chains of the two proteins were measured in both the free (unbound) and the complexed (bound) states using NMR spectroscopy. Furthermore, hydration dynamics were measured in water and in the confined space of a reverse micelle. The dynamic response observed leads to an unfavorable change in TΔSconf, with a more rigid, still hydrated interface. This comprehensive study of both protein and “ligand” (in this case, another protein) and the measured site-specific changes in dynamics and hydration sheds light on the thermodynamic contributions that enable fM affinities. Supported by grants from the NIH, The Mathers Foundation and NSF.