COUPLING NETWORKS AND SIDE-CHAIN DYNAMICS IN PROTEINS

生物信号通过分子通讯传递。虽然大量的注意力已经 针对表征蛋白质复合物中分子间通讯的结构基础， 已知信号如何在单个球状蛋白质或结构域内跨距离传播。等 事件发生在变构蛋白、分子识别组分、配体受体和酶中。 因此，跨距离的位点间通讯是蛋白质的一个基本特征。原子分辨率 需要对分子内通讯的研究来解释蛋白质的基于序列的调节 活性、底物特异性、配体调节和赋予耐药性的远端突变。的 远端残基对蛋白质活性位点影响对于蛋白质设计将是特别有价值的。的 本提案中的研究旨在确定站点间通信的机械基础， 生物物理和生物化学方法的组合。有三个具体目标针对中心假设 对于这个提议-侧链动力学介导蛋白质中的远程通信。实验将 Eglin C是一种小丝氨酸蛋白酶抑制剂，具有有利的特性， 提出的新方法。远程、成对通信或“耦合”的检测将是 从广泛的热力学双突变体循环分析。还将映射Couplin_ networks 通过侧链动力学的传播变化，如NMR H自旋弛豫测量所观察到的 在溶液中。为了测试信号传播的机制和通信网络的演变， 从设计角度来看，将进行“途径上”突变以调节位点之间的通讯。 与活性部位抑制环相连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。整个耦合网络的映射通过 所提出的方法是通用的，并且应该适用于其他蛋白质和蛋白质复合物。

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. Couplin_ 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.