COUPLING NETWORKS AND SIDE-CHAIN DYNAMICS IN PROTEINS

描述(申请人提供)：生物信号通过分子通讯传输。虽然人们对蛋白质复合体中分子间通信的结构基础进行了大量的研究，但对单个球状蛋白质或结构域中的信号如何跨距离传播却知之甚少。这样的事件发生在变构蛋白、分子识别组件、配体受体和酶中。因此，远距离的点对点通信是蛋白质的一个基本特征。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、底物专一性、配体调节和导致耐药的远端突变。远端残基对蛋白质活性部位的影响对蛋白质设计具有特别重要的意义。这项建议中的研究试图确定结合使用生物物理和生化方法的站点对站点通信的机制基础。三个具体的目标解决了这一提议的中心假设--侧链动力学调节蛋白质中的远程通讯。实验将在Eglin c上进行，Eglin c是一种小的丝氨酸蛋白酶抑制剂，具有良好的性能，以帮助开发所提出的新方法。对远距离、成对通信或“耦合”的检测将来自广泛的热力学双突变循环分析。耦合网络也将通过侧链动力学的传播变化来映射，如从溶液中的核磁共振氢自旋弛豫测量所观察到的。为了从设计的角度测试信号传播的机制和通信网络的演变，将进行“路径上”的突变，以调节站点之间的通信。与活性部位抑制环连接的通讯通路的功能意义将通过基于动力学的抑制试验进行评估。通过所提出的方法绘制整个偶联网络图是通用的，并且应该适用于其他蛋白质和蛋白质复合体。

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.