High-affinity protein complexes are critical to a large number of intricate regulatory processes. Their formation involves a complicated manifold of interactions that are diverse and complex. This complexity is reflected in the difficulty of computing the energetics of interactions between proteins using molecular structure alone. Indeed, the structure-based design of pharmaceuticals has been significantly impeded by this barrier. Understanding the fundamental origins of the energetics and dynamics of the interactions of proteins with both natural and pharmacological ligands is clearly critical to the optimization of "rational" drug design. Recent advances in nuclear magnetic resonance (NMR) relaxation methods have enabled the use of measures-of-motion between conformational states of a protein as a proxy for conformational entropy. There is now a strong indication from recent studies utilizing this approach that changes in conformational entropy can significantly influence the thermodynamics of the interaction of small molecule ligands with proteins. Therefore, we will examine this and related issues in the context of the ser/thr kinase p38?. p38? is intimately associated with a variety of disease states, including cancer and neurological diseases, and is an active target for pharmaceutical development. Experiments are proposed to examine the changes in fast internal motion in this protein upon interaction with both natural and pharmacological small molecule ligands. Advanced NMR relaxation methods will be employed to measure main chain and side chain motion. A variety of analytical strategies will be used to gain insight into the quantitative contributions to the thermodynamics of complex formation and to discover their structural origins. In addition, the dynamical effects of regulatory protein bindng will also be examined. These data will go to the heart of the physical mechanism for activation and deactivation of this critical kinase by both natural effector proteins and man-made molecules. Complementary hydrogen exchange studies will also be carried out with the goal of exposing cooperative interactions within p38?. This view will be particularly informative with respect to the emerging class of pseudo-allosteric drugs. A novel NMR-based approach using high-pressure perturbation and rapid three dimensional radial sampling will be employed to overcome limitations in the standard "native state" hydrogen exchange method in the context of large proteins, such as p38?. Overall, the proposal rests on a significant foundation of preliminary results including an unusually deep and robust library of resonance assignments for a ser/thr kinase.
The physical basis for the regulation of protein ser/thr kinases is key to understanding their role in cellular signaling in human biology and disease. This proposal seeks to test a fundamental hypothesis regarding the origin of high affinity interactions of proteins with proteins and drug-like ligands. Detailed knowledge of such interactions will significantly improve the cost effective rational design of pharmaceuticals.
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