T-4 Lysozyme as a Model for Protein Folding

这个建议的目的是为了更好地理解蛋白质结构、稳定性和动力学之间的相互关系，以T4溶菌酶为模型。这个由164个残基组成的中等大小的蛋白质具有已知的三维晶体结构。该蛋白有两个结构域，没有天然的二硫键。存在着许多调节其热力学稳定性的突变，俄勒冈大学布赖恩·马修斯的实验室已经确定了100多个突变晶体结构。我们用现代核磁共振技术对野生型和几种突变型蛋白质的酰胺共振进行了归属。我们建议使用这些方法来研究(1)蛋白质19个羧基的电离行为，以了解蛋白质中的静电和极性效应：(2)折叠状态的动力学；(3)确定由于对氢交换的保护而被检测到的早期折叠中间体在折叠路径中的作用，它提供了有限的酰胺；(4)完成了侧链共振的指定，目的是确定蛋白质的溶液结构。蛋白质的线性氨基酸序列决定了蛋白质在溶液中获得的复杂的三维形状。这项提案研究了如何利用线性信息将蛋白质折叠成其三维结构。我们将研究：(1)电荷相互作用在稳定折叠状态中的作用，(2)折叠状态的刚性，(3)随机的未折叠聚合物链转变为最终折叠状态的时间过程，以及(4)最终折叠状态的溶液结构。

The purpose of this proposal is to better understand the interrelationships of protein structure, stability, and dynamics using T4 lysozyme as a model. This moderate sized protein of 164 residues has a known 3-dimensional crystal structure. The protein has two structural domains and no natural disulfide bonds. Many mutants exist that modulate its thermodynamic stability and more than 100 mutant crystal structures have been determined by Brian Matthews' laboratory at the University of Oregon. We have assigned the amide resonances of the wild type and several mutant proteins using modern nuclear magnetic resonance techniques (NMR). We propose to use these methods to investigate (1) the ionization behavior of the 19 carboxyl groups of the protein with the goal of understanding electrostatic and polarity effects in the protein; (2) the dynamics of the folded state; (3) determine the role in the folding pathway of an early folding intermediate which was detected as a result of the protection to hydrogen exchange it afforded a limited set of amides; (4) complete the assignments of the sidechain resonances with the goal of determining the solution structure of the protein. %%% The linear amino acid sequence of a protein determines the complex three-dimensional shape that protein acquires in solution. This proposal investigates how that linear information is used to fold the protein into its three-dimensional structure. We will investigate: (1) the role of charge-charge interactions in stabilizing the folded state, (2) the rigidity of the folded state, (3) the time course of the conversion of a random, unfolded polymer chain into the final folded state, and (4) the structure, in solution, of the final folded state.