Protein Dynamics in Dihydrofolate Reductase Catalysis

描述(申请人提供)：蛋白质是动态的分子机器，在广泛的时间尺度上进行运动。虽然从理论和实验上都有相当多的证据表明许多酶具有内在的灵活性，但关于蛋白质动力学和酶催化之间的关系的基本问题仍然没有得到回答。蛋白质运动是否与化学转化有关，或者它们是否主要参与控制底物、产物或辅因子的流量？蛋白质运动在从预先组织的基态结构到促进化学反应的活性部位配置的过程中扮演什么角色？催化所需的活性中心构象变化的时间尺度是多少？酶的能量格局在催化循环中是如何调节的，在进化过程中又是如何形成的？是否存在与物种相关的蛋白质动力学差异和可用的构象亚态，这些差异可能被用于开发高选择性的药物，从而更好地区分来自人类和病原体的酶？这些问题将用最先进的核磁共振方法和多构象室温X射线结晶学来解决，以阐明一种特性特别好的酶--二氢叶酸还原酶(DHFR)的动态性质。DHFR是抗叶酸药物的靶标，如抗癌药物甲氨蝶呤以及抗菌药甲氧苄啶和伊卡普林。这项拟议的研究将使人们对这种重要酶的内在分子动力学以及它的运动如何在催化循环的不同阶段与底物、辅因子和产物相互作用产生新的理解。它还将为进化在塑造大肠杆菌和人类DHFR能量格局中的作用提供新的见解，并将促进我们对蛋白质动力学和催化功能之间的关系的理解。

DESCRIPTION (provided by applicant): Proteins are dynamic molecular machines, undergoing motions on a wide range of time scales. Although there is considerable evidence both from theory and experiment that many enzymes are inherently flexible, fundamental questions about the relationship between protein dynamics and enzyme catalysis remain unanswered. Are protein motions coupled to the chemical transformation, or are they involved primarily in controlling the flux of substrate, products, or cofactors? What role do protein motion play in progression from the preorganized ground state structure to an active site configuration that facilitates the chemical reaction? What is the time scale of active site conformational changes required for catalysis? How is the energy landscape of the enzyme modulated during the catalytic cycle and how is it shaped during evolution? Are there species-related differences in protein dynamics and available conformational substates that might potentially be exploited for development of highly selective drugs that better discriminate between enzymes from humans and pathogens? These issues will be addressed using state-of-the-art NMR methods and multi-conformer room temperature X-ray crystallography to elucidate the dynamic properties of an exceptionally well- characterized enzyme, dihydrofolate reductase (DHFR). DHFR is the target for anti-folate drugs such as the anticancer agent methotrexate and the antibacterials trimethoprim and iclaprim. The proposed research will lead to new understanding of the intrinsic molecular dynamics of this important enzyme and how its motions are modulated by interaction with substrate, cofactor, and products at various stages in the catalytic cycle. It will also provide novel insights into the role of evolution in shaping the energy landscapes of E. coli and human DHFR and will advance our understanding of the relationship between protein dynamics and catalytic function.