Protein Dynamics in Dihydrofolate Reductase Catalysis

蛋白质是动态的分子机器，在广泛的时间尺度上进行运动。 尽管来自理论和实验的大量证据表明，许多酶是 本质上是灵活的，这是一个基本的问题，即蛋白质波动如何，甚至是否会与 催化作用仍未得到回答。蛋白质运动是否与化学转化有关， 或者它们主要参与控制底物、产品或辅因子的流量？什么是 催化所需活性中心构象变化的时间尺度？能源形势如何？ 在催化循环中被调节的酶？这些问题和其他问题将通过以下方式解决 用最先进的核磁共振方法阐明一种特殊油井的动力学性质- 特征酶，来自大肠杆菌的二氢叶酸还原酶，在形成的所有中间状态下 沿着它的反应路径。DHFR是抗叶酸药物的靶点，如抗癌剂 甲氨蝶呤和甲氧苄啶的抗菌药具有重大的生物医学意义。这个 拟议的研究将集中在微秒-毫秒时间尺度波动的特征上 在活性中心，与DHFR催化中的关键事件具有相同的时间尺度。核磁共振实验 将提供活动场地的慢动作的详细的动力学和热力学描述 在催化循环中涉及的所有中间状态中的环，以及在精心挑选的突变体中 破坏催化过程。这些实验建立在该实验室早期的研究基础上 绘制了DHFR的活性中心构象状态及其快速(ps-ns)时间尺度动力学。这个 拟议的研究将为蛋白质运动和催化之间的耦合提供新的见解 在DHFR中的功能，以及了解这些运动是如何通过相互作用进行调制的 在催化循环的不同阶段有底物、辅因子和产物。

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, the fundamental question of how, or even if, protein fluctuations couple to catalytic function remains unanswered. Are protein motions coupled to the chemical transformation, or are they involved primarily in controlling the flux of substrate, products, or cofactors? 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? These and other issues will be addressed using state-of-the-art NMR methods to elucidate the dynamic properties of an exceptionally well- characterized enzyme, dihydrofolate reductase from E. coli, in all of the intermediate states formed along its reaction pathway. DHFR is the target for anti-folate drugs such as the anticancer agent methotrexate and the antibacterial trimethoprim and is of major biomedical significance. The proposed research will focus on characterization of microsecond-millisecond time scale fluctuations in the active site, on the same time scale as key events in DHFR catalysis. The NMR experiments will provide a detailed dynamic and thermodynamic description of slow motions of the active site loops in all intermediate states involved in the catalytic cycle and in carefully selected mutants that impair the catalytic process. These experiments build upon earlier research from this laboratory that mapped the active site conformational states of DHFR and its fast (ps-ns) time scale dynamics. The proposed research will provide novel insights into the coupling between protein motions and catalytic function in DHFR, as well as an understanding of how these motions are modulated by interaction with substrate, cofactor, and products at various stages in the catalytic cycle.