Molecular recognition by proteins is fundamental to almost every biological process, particularly the protein associations underlying cellular signal transduction. Understanding the basis for protein- protein interactions requires the full characterization of the thermodynamics of their association. Historically it has been virtually impossible to experimentally estimate changes in residual protein entropy, a potentially important component of the free energy of protein association. However, solution NMR spectroscopy has recently emerged as a powerful tool for characterizing the dynamics of proteins and has thereby gained access to their residual entropy. The initial view provided by NMR relaxation studies of the functional dependence of protein dynamics (entropy) in calmodulin is startling. The change in internal dynamics of calmodulin varies significantly upon binding a variety of target domains. Surprisingly, the apparent change in the corresponding residual entropy is linearly related to the overall binding entropy. These results indicate that changes in protein conformational entropy can contribute significantly to the free energy of protein-ligand association. It therefore seems evident that protein entropy can be exploited in the maturation of high affinity interactions either by biological evolution or by human intervention such as in the design of protein-targeted pharmaceuticals. This proposal seeks to more fully characterize the nature of the internal dynamics of proteins and to establish the degree to which the corresponding entropy contributes to the thermodynamics governing the binding of ligands. To achieve this, we will expand the library of NMR-based probes for internal motion and apply them in a variety of contexts. The interaction of calmodulin with calmodulin-binding domains of regulated proteins will continue to be a central model system. Calmodulin is critical to calcium-mediated signal transduction and is intimately involved in the regulation of critical physiological and cellular responses including control of smooth muscle contraction in the digestive tract. It interacts with and influences the activity of over 300 proteins. It is also a well-behaved system for NMR-based relaxation studies and promises to provide a wealth of insight into the nature and role of protein dynamics (entropy) in protein-ligand interactions. In addition, to assess whether the insights learned from the calmodulin system are general we will carry out similar investigations of other protein-ligand interactions. This will include the interaction of the Leukemia Inhibitory Factor and Oncostatin M cytokines with the GP130 receptor. GP130 it as the intersection of a number of important signaling networks of significant clinical interest, particularly in inflammatory bowel disease and cancer. In collaborative work, we will compare crystallographic analysis and molecular dynamics simulations with the experimental results obtained and undertake computational analyses of the thermodynamic origins of binding. Overall these studies promise to reveal novel insights into the physical origins of protein motion, their biological significance and will begin to illuminate the potential for their exploitation in the context of pseudo-allosteric drug inhibitors. [unreadable] [unreadable] [unreadable]
NIH ExPorter
NIH ExPorter