COUPLING NETWORKS AND SIDE-CHAIN DYNAMICS IN PROTEINS

描述（申请人提供）：生物信号是通过分子通信传输的。虽然大量的注意力集中在描述蛋白质复合体分子间通讯的结构基础上，但对于信号如何在单个球状蛋白或结构域内跨距离传播知之甚少。这些事件发生在变构蛋白、分子识别组分、配体受体和酶中。因此，跨距离的位点间交流是蛋白质的基本特征。需要分子内通讯的原子分辨率研究来解释基于序列的蛋白质活性调节、底物特异性、配体调节和赋予耐药性的远端突变。远端残基对蛋白质活性位点的影响对蛋白质设计特别有价值。本提案中的研究旨在利用生物物理和生化方法的结合来确定位点-位点通信的机制基础。三个具体的目标解决了这一提议的中心假设-侧链动力学介导蛋白质的远程通信。实验将在eglin c上进行，eglin c是一种小型丝氨酸蛋白酶抑制剂，具有良好的特性，有助于开发所提出的新方法。远距离、成对通信或“耦合”的检测将通过广泛的热力学双突变循环分析来实现。耦合网络也将通过侧链动力学的传播变化来绘制，正如从溶液中的NMR H自旋弛豫测量中观察到的那样。为了从设计的角度测试信号传播机制和通信网络的进化，将进行“通路上”突变，以调节位点之间的通信。与活性位点抑制环连接的通信通路的功能意义将通过基于动力学的抑制试验进行评估。通过所提出的方法映射整个耦合网络是通用的，应该适用于其他蛋白质和蛋白质复合物。

DESCRIPTION (provided by applicant): Biological signals are transmitted via molecular communication. While a great deal of attention has been directed at characterizing the structural basis of intermolecular communication in protein complexes, little is known about how signals propagate across distances within individual globular proteins or domains. Such events occur in allosteric proteins, components of molecular recognition, ligand receptors, and enzymes. Site-site communication across distances is therefore a fundamental feature of proteins. Atomic resolution studies of intramolecular communication are needed to explain sequence-based modulations of protein activity, substrate specificity, ligand regulation, and distal mutations conferring drug resistance. The influence of distal residues on a protein's active site will be particularly valuable for protein design. The research in this proposal seeks to determine the mechanistic basis for site-site communication using a combination of biophysical and biochemical approaches. Three specific aims address the central hypothesis for this proposal - that side-chain dynamics mediate long-range communication in proteins. Experiments will be carried out on eglin c, a small serine protease inhibitor possessing favorable properties to aid development of the proposed novel approaches. Detection of long-range, pair-wise communication, or "coupling", will be made from extensive thermodynamic double-mutant cycle analysis. Coupling networks will also be mapped through propagated changes in side-chain dynamics, as observed from NMR H spin relaxation measurements in solution. To test mechanisms of signal propagation and the evolution of communication networks from a design perspective, "on-pathway" mutations will be made in order to modulate communication between sites. The functional significance of communication pathways that connect with the active site inhibitory loop will be assessed from a kinetics-based inhibition assay. The mapping of entire coupling networks through the approach presented is general and should be applicable to other proteins and protein complexes.