Our specific aims are twofold, to continue our studies on the mechanism of protein hydrogen exchange, and to determine the relationship between internal mobility and thermodynamic stability of various submolecular domains of BPTI. These are highly interrelated; conclusions from each area of study have a direct bearing on the interpretation of results from the other. Hydrogen exchange in proteins refers to the isotype exchange kinetics of a protein NH hydrogen with solvent hydrogen. The exchange rate is a function of internal protein motions that expose the exchanging NH to solvent. Historically, hydrogen exchange provided the first experimental insight into the dynamical aspect of protein structure. We measure exchange rates of assigned NH protons by 1H NMR. OUr lab has produced a number of significant contributions to the basic understanding of the mechanism of hydrogen exchange in proteins. We propose to continue this avenue of inquiry, using the model protein bovine pancreatic trypsin inhibitor (BPTI). In addition, we are probing the relationship between protein dynamic structure and protein thermodynamic stability. 1) The mechanism of hydrogen exchange in proteins involves two central question. What structural factors account for slowing of exchange rates in proteins compared to equivalent extended peptides? What kinds of conformational fluctuations lead to the exposure of buried NH groups to solvent when the protein is in the folded state? We study these questions by determining the basic kinetics of the exchange reaction, namely, the acid and base exchange rate constants, and their variation with temperature, pH and ionic strength for BPTI in solution and in crystals. We use BPTI variants obtained by site directed mutagenesis to probe the basic chemical and conformational mechanism of protein hydrogen exchange. 2) What is the relationship between protein internal mobility and thermodynamic stability? Submolecular packing domains, or microdomains, have been defined based on hydrogen exchange properties. These are the "knot", "matrix" and "surface". The knot is the rigid hydrophobic core of atoms that pack the very slowest exchanging protons. The matrix is composed of the more flexible regions of the buried interior containing more rapidly exchanging buried NH's. The matrix in BPTI, we find, had two components, a) the matrix loops comprised of long, overlapping aperiodic segments enclosing buried water molecules, and b) a hinge region between the core and the matrix. The surface contains the NH's that are exposed to solvent and not intramolecularly H-bonded. We use site-directed mutagenesis to selectively perturb the core, matrix, or surface of BPTI. Then we measure changes in internal flexibility by hydrogen exchange and proteolytic susceptibility. The thermodynamics of the cooperative denaturation are determined by calorimetry and circular dichroism. Time average structures of mutant BPTI's are modeled using NMr distances and constrained molecular dynamics. With these approaches we ask a number of questions regarding the relationship between protein dynamics and thermodynamics.