A DYNAMIC SIMULATION OF LIGAND BINDING TO DHF REDUCTASE

我们建议继续我们对结构力学、动力学的研究 和二氢叶酸还原酶(DHFR)-配体家族的能量学 系统。长期目标包括理解这些 基本的生物过程，如生物识别、酶 特异性、调节和蛋白质结构。我们已经取得了重大进展 了解配体与还原酶结合的研究进展。我们的 计算机仿真软件已在Cyber205上实现 一台超级计算机，使我们能够对一个 系统的大小与DHFR相同，细节和严谨程度与小型 分子。我们对三个系统进行了广泛的研究，其中E。 ColiDHFR与抗生素甲氧苄氨嘧啶(TMP)的二元复合体， 三元络合物，DHFR-TMP-NADPH，和鸡肝三元络合物。 初步分析了这些系统的结构和能量学 增加了对辅因子(NADPH)的协作性的理解 和抑制物(TMP)结合在细菌酶中，缺乏这一点 脊椎动物酶中的现象，以及TMP与 细菌酶。对这种药物受体系统的详细分析是 还在继续。 我们带来了一系列理论工具来解决这些问题。 我们现在建议进行广泛的分子动力学模拟 晶体环境中的几个DHFR络合物。这将是第一次 动力学、结构、温度因子和时间的计算 环境中酶复合体的平均电子密度 对这些特性进行了测量，使严格的比较成为可能。 与加州大学的Kraut博士和Matthews博士一起，我们将使用这些X光 结构来测试和验证模拟结果。本节目 关键依赖于与实验者的密切合作； 约瑟夫·克劳特教授，他正在对 各种还原酶复合体的X射线结构，以及Kraut和 约翰·阿贝尔森在他的实验室里进行了各种定点突变 DHFR的实验正在进行中。 还原酶X射线结构计算值与实测值的比较 将验证理论处理的结果并揭示 可能存在的差异，需要进一步了解和 更正。这一阶段的完成将导致对 这些系统处于运行的原子间作用力、能量学的水平 而动力学决定了它们的性质。 最后，我们提出了模拟伴随键的结构变化 DHFR氨基酸序列的变化，目的是了解 蛋白质结构，并将所需的功能引入其中 蛋白。这些模拟将通过设计的产品进行测试 酶的定点突变和生物活性的评价。

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.