A DYNAMIC SIMULATION OF LIGAND BINDING TO DHF REDUCTASE

我们建议继续研究结构力学、动力学 二氢叶酸还原酶（DHFR）-配体家族及其能量学 系统. 长期目标包括理解这些 基本的生物学过程，如生物识别，酶 特异性、调节和蛋白质结构。 我们取得了重大 进一步了解配体结合还原酶。 我们 计算机模拟软件已经在Cyber 205上实现 超级计算机，使我们能够进行广泛的模拟研究， 系统的大小DHFR具有相同的细节和严谨性，适用于小 分子。 我们对三个系统进行了广泛的研究，E。 大肠杆菌DHFR与抗生素甲氧苄啶（TMP）的二元复合物， 三元复合物、DHFR-TMP-NADPH和鸡肝三元复合物。 对这些系统的结构和能量学进行了初步分析， 增加了对辅酶（NADPH）协同作用的理解 和抑制剂（TMP）结合在细菌酶中，缺乏这种 现象的脊椎动物酶，并加强结合TMP的 细菌酶 对这种药物受体系统的详细分析是 继续。 我们带来了一系列的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， 几个DHFR复合物在晶体环境中。 这将是第一 动力学、结构、温度因素和时间的计算 酶复合物的平均电子密度的环境中， 测量了这些性能，使得严格的比较成为可能。 我们会和加州大学圣地亚哥分校的克劳特博士和马修斯博士一起用这些X光片 结构来测试和验证模拟的结果。 这个程序 关键取决于与实验者的密切合作; 约瑟夫·克劳特教授正在对 结构的各种还原酶复合物的x射线，教授克劳特和 约翰·阿贝尔森，在他的实验室里， DHFR的实验正在进行中。 还原酶X射线结构的计算与观测比较 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， 纠正一下 完成这一阶段将导致理解 这些系统在原子间相互作用力的水平上， 和动力学决定了它们的性质。 最后，我们建议模拟结构变化伴随着关键 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 这些模拟将通过生产设计的 酶的位点特异性诱变和生物活性的评估。

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.