The fundamental premise of this proposal is that proteins are dynamic entities, that their internal motion is an expression of an inherent and significant amount of conformational entropy and that changes in conformation entropy can greatly influence the energetics of protein function. Below we will summarize recent results that support these assertions. Perhaps the simplest functional context is the binding of a ligand by a protein. Thus our general hypothesis is that the thermodynamics of protein-ligand interactions can be influenced via changes in the protein internal entropy. Though this idea has been in the literature for some time it is only recently that the experimental methods that are capable of illuminating it have become available. We will test the generality of a role for protein dynamics (and the entropy it represents) in molecular recognition by proteins and in the more sophisticated allosteric response. The former will build upon an existing array of examples that indicate a general roughly linear relationship between the contributions of a change in conformational entropy upon ligand binding and the overall binding entropy. Though such a relationship is not required its existence suggests that evolution has exploited conformational entropy in the optimization of protein-ligand thermodynamics. This idea will be quantitatively explored using the recently calibrated "entropy meter" that relies on a dynamical proxy for conformational entropy. Comprehensive measurements of internal protein motion will be undertaken using the now well-established solution NMR relaxation methods. In an effort to more fully understand the propagation of dynamics within the protein matrix, a high-pressure perturbation study of protein motion will be carried out to illuminate coupling of motion. Recent work by others on the catabolite activator protein suggests that conformational entropy may play a central role in protein-DNA recognition. We therefore propose to examine the structural and dynamic properties underlying protein-DNA recognition in the lac repressor. The lac repressor is a paradigm for genetic regulation in prokaryotes. Overall these studies will greatly expand our appreciation of the nature of protein motion and the role of the conformational entropy that it represents in the energetics of protein function.
The physical basis for formation of high affinity complexes involving proteins is key to understanding their role in cellular signaling in human biology and disease. This proposal seeks to test a fundamental hypothesis regarding the contribution of internal protein conformation entropy to the energetics of high affinity interactions involving proteins. Detailed knowledge of such interactions will greatly enhance our understanding of the origins and role of complex biochemical signaling and may also significantly improve the cost effective rational design of pharmaceuticals directed at protein targets.
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|Moorman, Veronica R; Valentine, Kathleen G; Bédard, Sabrina et al. (2014) Dynamic and thermodynamic response of the Ras protein Cdc42Hs upon association with the effector domain of PAK3. J Mol Biol 426:3520-38|
|Sharp, Kim A; Kasinath, Vignesh; Wand, A Joshua (2014) Banding of NMR-derived methyl order parameters: implications for protein dynamics. Proteins 82:2106-17|
|Wand, A Joshua; Moorman, Veronica R; Harpole, Kyle W (2013) A surprising role for conformational entropy in protein function. Top Curr Chem 337:69-94|
|Kasinath, Vignesh; Sharp, Kim A; Wand, A Joshua (2013) Microscopic insights into the NMR relaxation-based protein conformational entropy meter. J Am Chem Soc 135:15092-100|
|Kasinath, Vignesh; Valentine, Kathleen G; Wand, A Joshua (2013) A 13C labeling strategy reveals a range of aromatic side chain motion in calmodulin. J Am Chem Soc 135:9560-3|
|Wand, A Joshua (2013) The dark energy of proteins comes to light: conformational entropy and its role in protein function revealed by NMR relaxation. Curr Opin Struct Biol 23:75-81|
|Fu, Yinan; Wand, A Joshua (2013) Partial alignment and measurement of residual dipolar couplings of proteins under high hydrostatic pressure. J Biomol NMR 56:353-7|