We propose to continue our studies of the structural mechanics, dynamics and energetics of a family of dihydrofolate reductase (DHFR) - ligand systems. The long range goals involve the understanding of such fundamental biological processes as biological recognition, enzyme specificity, regulation and protein architecture. We have made significant progress toward an understanding of ligand binding to reductase. Our computer simulation software has been implemented on a Cyber 205 super-computer allowing us to carry out extensive simulation studies on a system the size of DHFR with the same detail and rigor as applied to small molecules. We have carried out extensive studies of three systems, the E. coli binary complex of DHFR with the antibiotic trimethoprim (TMP), the ternary complex, DHFR-TMP-NADPH, and the chicken liver ternary complex. Preliminary analysis of the structure and energetics of these systems has led to an increased understanding of the cooperativity of cofactor (NADPH) and inhibitor (TMP) binding in the bacterial enzyme, the lack of this phenomenon in the vertebrate enzyme, and the enhanced binding of TMP to the bacterial enzyme. Detailed analysis of this drug-receptor system is continuing. We have brought an array of theoretical tools to bear on these problems. We now propose to carry out extensive molecular dynamic simulations of several DHFR complexes in the crystal environment. This will be the first computation of the dynamics, structure, temperature factors and time averaged electron density of enzyme complexes in the environment in which these properties were measured, making a rigorous comparison possible. Together with Drs. Kraut and Matthews of UCSD, we'll use these x-ray structures to test and verify the results of the simulation. This program depends critically on the close collaboration with experimentalists; Professor Joseph Kraut, who is carrying out extensive study of the structure of various reductase complexes by x-ray, and Professors Kraut and John Abelson, in whose laboratory a variety of site specific mutagenesis experiments of DHFR are taking place. Comparison of the calculated and observed x-ray structures of reductase will verify the results of the theoretical treatment and reveal discrepancies which may exist and require further understanding and correction. Completion of this stage will lead to an understanding of these systems at the level of the operative interatomic forces, energetics and dynamics determining their properties. Finally, we propose to simulate the structural changes accompanying key changes in the amino acid sequence of DHFR, with the goal of understanding protein architecture, and introducing desired functionalities into this protein. These simulations will be tested by production of the designed enzyme by site specific mutagenesis and assessment of biological activity.
