Protein dynamics from femtoseconds to milliseconds as crafted by natural and laboratory evolution: towards enzyme design

这个R35应用程序描述了我们持续和扩展的计划，以开发和应用计算 方法来研究蛋白质动力学如何在许多时间尺度上有助于酶催化，以及如何一些 酶是通过进化来制造的，以利用多个时间尺度上的蛋白质动力学。这些知识将 最终为设计人造酶提供信息-这是一个巨大的挑战，但尚未得到满足。 我们对酶催化的研究始于几年前的第一次应用过渡路径采样 酶的化学反应。反应轨迹系综的生成沿着与反应 坐标识别允许我们假设促进振动的蛋白质的概念：快速蛋白质 在活性位点处或附近的动力学，其直接耦合到越过过渡态势垒的通道， 反应这种运动在多种酶系统中被发现（但不是全部），并且它们的重要性得到了验证 实验合作者。最近，将这些方法应用于人工酶， 经过实验室进化的优化，已经表明进化过程引入了这样的运动 我们已经确定了蛋白质结构的变化，允许创建和耦合 动力通过从头静态结构方法创建的“Theozymes”取得了有限的成功，而 实验室的进化已经允许这些蛋白质发展出显著的催化能力， 我们将继续和显着扩大我们的计划在这一具有挑战性的主题，通过两个扩展 所采用的方法，并应用于天然和实验室进化的酶家族。在 除了了解蛋白质进化如何导致快速动力学与屏障通过的耦合，我们 将扩展我们的方法，以便能够绘制出在时间上距离穿越地球更远的运动。 化学屏障也可以是功能的中心。我们还将开发方法，证明如何快速 运动可以使系统准备毫秒级的构象变化。我们的计划目标是 了解蛋白质动力学如何从亚皮秒促进振动到微秒域 运动到毫秒构象运动可能是相互关联的，并有助于形成酶功能， 这些运动是如何被进化所创造的结构所协调的。 这些工具的发展与实验室和自然进化的酶的研究相结合 家庭将允许隔离的“动态工具箱”所采用的选择。我们已经找到了如何在 一个实验室进化出的酶，在关键位置引入和失去氢键， 创造一个促进振动;成功完成拟议的计划将扩大这种 研究了适合催化周转的蛋白质运动时间尺度的全范围，沿着 在蛋白质催化剂中产生这种动力学所需的结构变化。

This R35 application describes our continuing and expanding program to develop and apply computational methods to study how protein dynamics on many timescales contributes to enzymatic catalysis, and how some enzymes are crafted by evolution to make use of protein dynamics on multiple timescales. This knowledge will eventually inform approaches to design artificial enzymes – a grand challenge, as yet unmet. Our studies of enzymatic catalysis began years ago with the first application of Transition Path Sampling to chemical reaction in enzymes. The generation of reactive trajectory ensembles along with reaction coordinate identification allowed us to postulate the concept of the protein promoting vibration: rapid protein dynamics at or near the active site that are directly coupled to passage over the transition state barrier to reaction. Such motions were found in multiple enzyme systems (but not all,) and their importance was verified by experimental collaborators. More recently, application of these methods to artificial enzymes that are subjected to optimization by laboratory evolution has shown the evolutionary process introduces such motion into a static design and we have identified protein structural changes that allows the creation and coupling of the dynamics. “Theozymes” created by de novo static structural methods have had limited success, while laboratory evolution has allowed these proteins to develop significant catalytic power, We will continue and significantly expand our program on this challenging topic through extensions of both methodologies employed and with application to both natural and laboratory evolved enzyme families. In addition to understanding how protein evolution leads to the coupling of rapid dynamics to barrier passage, we will extend our approaches to be able to map how motions far more remote in time from the passage over the chemical barrier can also be central to function. We will also develop methods that demonstrate how rapid motions can prime the system for millisecond conformational change. Our goals for the program are to understand how protein dynamics ranging from sub picosecond promoting vibrations to microsecond domain motions to millisecond conformational motion are potentially inter-related and help form enzyme function, and how such motions are orchestrated by the structure crafted by evolution. The development of such tools coupled with studies of both laboratory and naturally evolved enzyme families will allow the isolation of a “dynamics toolbox” employed by selection. We have already found how in one laboratory evolved enzyme the introduction and loss of hydrogen bonds in strategic locations results in the creation of a promoting vibration; successful completion of the proposed program will expand such investigation to the full range of protein motion timescales appropriate to catalytic turnover, along with the architectural changes needed to create such dynamics in the protein catalyst.