Molecular Basis of Stability and Folding of Proteins

Johns Hopkins University, Baltimore MD

Investigators

Abstract

The goals of this research program are to further understanding of electrostatic contributions to the stability and function of proteins. Electrostatics is the most useful correlator of structure and function in biological macromolecules, especially in proteins. Without deep understanding of the contributions of electrostatics, the computational methods that are needed to bridge the gap between structure and energy of proteins cannot be improved, nor can the structural basis of stability and function of proteins be established. To accomplish these goals, two sets of experimental studies will be carried out. One set will address the relationship between the dynamic character of proteins and their electrostatic properties. To understand how local structural fluctuations modulate pKa values, the pKa of Asp and Glu residues in staphylococcal nuclease will be measured by NMR spectroscopy. The pKa values will also be used to analyze the acid unfolding of nuclease with an ensemble-based computational method that has the potential to describe a ligand-linked structural transition at unprecedented level of detail. Global conformational changes will be studied by measurement of the Stokes radii of the native and denatured ensemble with size-exclusion chromatography. The goal of the second set of studies is to elucidate the determinants of the energetics of short-range electrostatic interactions. A cluster of ionizable and polar residues in staphylococcal nuclease will be studied systematically. The approach entails perturbation of interactions with mutagenesis, crystallography to obtain structures, NMR to measure pKa values, and measurements of stability by chemical and temperature denaturation. A central goal of this research is to develop computational tools for structure-based calculation of electrostatic energy and pKa values. The experimental studies will contribute the physical insight needed to guide the improvement of computational models, and provide the data needed to test these methods. To the extent that the concepts and computational tools that emerge from these studies have a direct bearing on problems in protein engineering, in rational, structure-driven design, and in biotechnology, they will have a deep and broad impact on society.