Project 2 is concerned with the elucidation of the structure and dynamics of a metallo-beta-lactamase from a clinically important anaerobic organism, Bacteroides fragilis. The goals of the project are two-fold, to obtain information on a molecule that is a key component of antibiotic resistance in the organism, and to utilize this system as one of the paradigms to investigate coupling between polypeptide chain dynamics and enzymatic function. Since NMR is the premier method for obtaining site- specific information on polypeptide dynamics in solution, the focus of the project is a extensive NMR study of the B. fragilis metallo-beta- lactamase, the wild type enzyme free and in complex with an inhibitor and mutant proteins specifically designed on the basis of the kinetic, structural and dynamic information available on the wild-type protein. Complete resonance assignments will be made for the active form of the enzyme, which contains two zinc ions. Preliminary data are excellent, and indicate that such assignments will be possible using a combination of Calpha and CO-based strategies. Complete resonance assignments will also be made for a complex of the enzyme with a substrate-analog, and a solution structure determination will be made for this complex. Polypeptide dynamics will be investigated by NMR for both the free enzyme an the complex, using 15N, 13C and 2H relaxation measurements. The study will provide the basis for mutagenesis of the metallo-beta-lactamase (Project 3) and for the elucidation of the reaction pathway (Project 4). The fourth specific aim concerns the study of local structure in the active site of the enzyme, using cadmium-substituted metallo-beta- lactamase. The identity and bonding of the ligands and the solvent exposure of the metal site can be obtained from these measurements, together with an estimate of the dissociation constants of the metal ions. The final specific aim will follow from the interactions between the components of the Program Project. Resonance assignments and relaxation measurements will be made for mutants designed on the basis of accumulated information form wild-type dynamics (Project 2), kinetic studies (Project 3) and calculations (Project 4). Structures will be determined for selected mutants either in solution by NMR or by X-ray crystallography by a collaborator, Dr. Osnat Herzberg. Information obtained from the synergistic interaction between the components of the Program Project should provide important insights into the role of dynamics in enzyme action.