Protein Phosphorylation as a Biophysical Switch: Structural, Dynamic and Thermodynamic Responses to Phosphorylation

Cornell University, Ithaca NY

Investigators

Abstract

Nature has designed a broad array of protein-based molecular switches that direct and control the flow of information, energy, and molecular cargo through living cells. One of the most important questions regarding functional switching of proteins is how the covalent attachment of a phosphate group so dramatically alters activity. The overall goal of this research is to elucidate biophysical mechanisms by which specific proteins are regulated by phosphorylation. Two model systems will be investigated, moesin and the c-Src SH3 domain. Moesin is a member of the ERM family of proteins that are involved in dynamic interactions with the actin cytoskeleton and the plasma membrane. SH3 domains are small, modular binding domains that mediate protein-protein interactions through binding to proline-rich sequence motifs. The moesin model system employed in this research consists of two interacting domains, N-FERM and C-ERMAD that comprise the N/C complex. Formation of the N/C complex prevents interactions between moesin and its cellular binding partners, and is regulated by phopshorylation of residue T558 in C-ERMAD and by PIP2 binding to N-FERM. This model system represents a "regulatory rheostat" where phosphorylation is coupled with additional regulatory factors to elicit variable activation. In a second model system, the ligand binding function of the c-Src SH3 domain is regulated by phosphorylation of residue Y57. This represents a prototype molecular switch where phosphorylation of a residue in or near the binding surface alters specificity and affinity of a modular binding domain. For each of the two model systems, multidimensional NMR spectroscopy will be used to observe phosphorylation-induced changes in structure, stability and dynamics, and isothermal titration calorimetry (ITC) for quantifying phosphorylation-induced changes in function. The biophysical mechanism by which each protein is regulated will be determined through amide exchange measurements, backbone and side chain dynamics studies, and determination of macroscopic and site-specific pKas. These investigations will provide a structural, dynamic and thermodynamic framework for understanding molecular switching mechanisms that have not yet been elucidated. Reversible protein phosphorylation is a ubiquitous mechanism utilized by all cells to switch protein function on and off. In order to visualize how protein phosphorylation regulates function, the structure, dynamics and thermodynamics of proteins in both phosphorylated and unphosphorylated states must be examined. Although a few detailed mechanisms are now characterized, our understanding of the varied mechanisms by which regulation by phosphorylation is accomplished is far from complete. This project aims to determine the biophysical mechanisms by which two particular proteins are regulated by phosphorylation. Coupling of the thermodynamic and site-specific perspectives obtained from ITC and NMR, respectively, will yield valuable insights into potential origins of binding energy, and the mechanisms used to alter this energy in biological molecular switches. These studies will advance our understanding of how biological processes are regulated by phosphorylation, and will impact important areas of research such as signal transduction, intracellular trafficking, and regulation of membrane structure. The research objectives will be accomplished primarily through the participation of graduate and undergraduate students who will receive education and training in molecular biology, protein chemistry, physical chemistry, computer technology, and multidimensional NMR spectroscopy.