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The interactions of biological macromolecules with water are fundamental to their structure, dynamics and function. Historically, characterization of the location and residence times of hydration waters of proteins in solution has been quite difficult. Solution NMR has long held promise but has been severely plagued by seemingly insurmountable problems. Confinement within the nanoscale interior of a reverse micelle slows water dynamics, allowing detection of global protein-water interactions using nuclear magnetic resonance techniques. Complications that normally arise from hydrogen exchange and long-range dipolar coupling are overcome by the nature of the reverse micelle medium.

Characterization of the hydration of ubiquitin demonstrates that encapsulation within a reverse micelle allows detection of dozens of hydration waters. Comparison of nuclear Overhauser effects obtained in the laboratory and rotating frames indicate a considerable range of hydration water dynamics is present on the protein surface. In addition, an unprecedented clustering of different hydration dynamic classes of sites is evident.

Nathaniel V. Nucci, Maxim S. Pometun, and A. Joshua Wand (2011) Site-resolved measurement of water-protein interactions by solution NMR. Nat. Struct. Mol. Biol. 18:245-224.

The physical basis for high affinity interactions involving proteins is complex and potentially involves a range of energetic contributions. Among these are changes in protein conformational entropy, which cannot yet be reliably computed from molecular structures. We have recently employed changes in conformational dynamics as a proxy for changes in conformational entropy of calmodulin upon association with domains from regulated proteins. The apparent change in conformational entropy was linearly related to the overall binding entropy. This view warranted a more quantitative foundation. Here we calibrate the “entropy meter” employing an experimental dynamical proxy based on NMR relaxation and show that changes in the conformational entropy of calmodulin are a significant component of the energetics of binding. Furthermore, the distribution of motion at the interface between the target domain and calmodulin are surprisingly non-complementary. These observations promote modification of our understanding of the energetics of protein-ligand interactions. This result opens the door to a broader examination of the role of conformational entropy in a range of protein-based phenomena.

Michael S. Marlow, Jakob Dogan, Kendra K. Frederick, Kathleen G. Valentine, and A. Joshua Wand (2010) The role of conformational entropy in molecular recognition by calmodulin. Nature Chemical Biology 6:352-358 (2010)