Structural and Mechanistic Studies of Essential Microbial Kinases

静电现象在生物过程中普遍存在，例如蛋白质折叠、结合和生物降解。 催化作用我们目前关于静电对蛋白质稳定性影响的知识主要来自于蛋白质 基于静态结构的泊松-玻尔兹曼工程实验和理论研究 计算。然而，虽然宏观测量通常不能将静电效应与 其他人，理论预测的准确性是有限的，缺乏明确的处理蛋白质介电 响应、构象动力学和由于未折叠状态下的残留结构的影响。因此，在本发明中， 尽管经过二十年的研究，诸如静电如何以及在多大程度上 调节蛋白质稳定性相互作用还没有得到充分的回答。缺乏准确的手段， 预测静电贡献不仅阻碍了对蛋白质稳定性的基本理解， 为推进计算蛋白质设计设置了障碍。本申请的目的是 1)通过进一步开发连续恒定pH，推进原子水平上的pH依赖现象研究 分子动力学和相关的方法，2）提高定量预测和详细的 通过研究几种模型系统，包括 核糖体L9蛋白的N-末端结构域、绒毛蛋白头片段亚结构域、亮氨酸拉链和内消旋、热配体 外周亚基结合结构域的超嗜热变体。拟议方法的发展 将为结构生物学社区提供强大的工具，用于研究广泛的静电 生物学中的现象在应用研究中获得的见解预计将改变以本土为中心的 蛋白质稳定性和功能的研究范式，并转变基于静态结构的蛋白质观 静电学他们还将帮助建立计算蛋白质设计的一般原则。

Electrostatic phenomena are ubiquitous in biological processes such as protein folding, binding, and catalysis. Our current knowledge of electrostatic effects on protein stability is mainly derived from protein engineering experiments and theoretical studies using static-structure based Poisson-Boltzmann calculations. However, while macroscopic measurements often cannot isolate electrostatic effects from others, the accuracy of theoretical predictions is limited by the lack of explicit treatment of protein dielectric response, conformational dynamics and effects due to residual structures in the unfolded state. As a result, despite two decades of research, important questions such as how and to what extent electrostatic interactions modulate protein stability have not been adequately answered. The lack of accurate means to predict electrostatic contributions not only hampers fundamental understanding of protein stability but also poses a roadblock for advancing computational protein design. The objectives of this application are to 1) advance atomic-level studies of pH-dependent phenomena by further developing continuous constant pH molecular dynamics and related methodologies, and 2) improve quantitative prediction and detailed understanding of electrostatic modulation of protein stability by studying several model systems including the N-terminal domain of ribosomal L9 protein, villin headpiece subdomain, leucine zipper, and meso-, thermoand hyper thermophilic variants of peripheral subunit binding domain. The proposed method development will provide the structural biology community with powerful tools for studying a wide range of electrostatic phenomena in biology. The insights gained in the application studies are expected to shift the native-centric paradigm of protein stability and function and transform the static-structure based view of protein electrostatics. They will also help establish general principles for computational protein design.