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

此R35应用程序描述了我们继续和扩展的计划，以开发和应用计算 方法研究多个时间尺度上的蛋白质动力学如何对酶催化做出贡献，以及一些 酶是由进化来制造的，以利用多个时间尺度上的蛋白质动力学。这一知识将会 最终提供设计人造酶的方法--这是一个尚未满足的巨大挑战。 我们对酶催化的研究早在几年前就开始了，第一次应用了过渡路径采样 酶中的化学反应。反应轨迹系综随反应的产生 坐标识别使我们得以假定促进振动的蛋白质的概念：快速蛋白质 在活性中心或活性中心附近的动力学，直接耦合到通过过渡态势垒 反应。这样的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 由实验合作者完成。最近，这些方法在人造酶上的应用 经过实验室优化的进化表明，进化过程引入了这样的运动 转化为静态设计，我们已经确定了蛋白质结构的变化，允许创建和偶联 动态平衡。用从头开始的静力结构方法创造的“Theozymes”成功有限，而 实验室的进化使这些蛋白质产生了显著的催化能力， 我们将继续并显著扩展我们关于这一具有挑战性的主题的计划，通过扩展这两个 所采用的方法和应用于天然和实验室进化的酶家族。在……里面 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，以便能够绘制出运动在时间上距离 化学屏障也可能是发挥作用的中心。我们还将开发一些方法来证明它有多快 运动可以为系统的毫秒构象变化做好准备。我们这个项目的目标是 了解从亚皮秒促进振动到微秒结构域的蛋白质动力学 运动到毫秒构象运动是潜在的相互关联的，并有助于形成酶功能，以及 这样的运动是如何由进化形成的结构来编排的。 这些工具的开发与实验室和自然进化酶的研究相结合 家庭将允许隔离选择所使用的“动力学工具箱”。我们已经了解了如何在 一种实验室进化的酶，在关键位置氢键的引入和丢失导致 创建促进振动；拟议计划的成功完成将扩大这样的 对适用于催化周转的全范围蛋白质运动时间尺度的研究，以及 在蛋白质催化剂中创造这样的动力需要结构上的改变。

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.