SYNTHETIC STUDIES OF PROTEIN STABILITY AND FOLDING

该合作研究计划将肽和小蛋白合成的最先进的温和化学方法与生物化学和生物物理研究相结合，以解决基本问题并开发有关蛋白质折叠，稳定性和动力学的假设。母体小球状蛋白靶点是牛胰胰蛋白酶抑制剂（BPTI），58个残基，具有三个二硫键，以及链球菌蛋白G（GB 1）的免疫球蛋白结合结构域，56个残基，无二硫键。在支持的第一阶段期间，显示出BPTI中的胱氨酸交联被成对的α-氨基-正丁酸（Abu）电子等排体取代提供了蛋白质，其在温和条件下呈现与在蛋白质折叠过程的前10-20毫秒期间形成的瞬时中间体的集合非常相似的结构。早期的发现为本提案的中心假设提供了基础：在球状蛋白中，可以识别核心基序，并且它们的元件可以在合适的肽中组合以构建天然样模块。设计的肽，长度约15-50个氨基酸残基，由通过天然或设计的序列连接的核心元件（来自BPTI和/或GB 1）组成，并且它们含有策略性放置的交联以将构象空间限制为更塌陷的构象。交联的设计理念是其主要功能是限制链的流动性（熵），而不是稳定折叠结构。核心模块本身具有很大的兴趣，并且建议表征它们的构象集合并优化它们的天然构象的稳定性。此外，已经开发了将核心模块串联连接以构建具有真正天然状态的较大蛋白质的策略。在整个研究过程中，持续改进和优化化学和纯化工具，以有效制备均质小蛋白质类似物，包括有效创建二硫化物，硫醚，侧链内酰胺和头-尾内酰胺交联桥所涉及的问题。正在进行的和拟议中的研究提供了新的方法，并导致显着的和普遍的见解，有助于“蛋白质折叠问题。"

This collaborative research program combines state-of-the-art mild chemical methods for peptide and small protein synthesis with biochemical and biophysical studies in order to address fundamental questions and develop hypotheses about protein folding, stability, and dynamics. The parent small globular protein targets are bovine pancreatic trypsin inhibitor (BPTI), 58 residues with an array of three disulfide bridges, and the immunoglobulin binding domain of streptococcal protein G (GB1), 56 residues without disulfides. During the first period of support, it was shown that replacement of cystine crosslinks in BPTI by paired alpha-amino-n-butyric acid (Abu) isosteres provides proteins which, under mild conditions, assume structures very similar to the ensemble of transient intermediates formed during the first 10-20 msec of the protein folding process. The earlier findings provide the underpinnings for the central hypothesis of the present proposal: in globular proteins, core motifs can be identified, and their elements can be combined in suitable peptides to construct native-like modules. The designed peptides, approximately 15-50 amino acid residues in length, consist of core elements (from BPTI and/or GB1) linked by natural or designed sequences, and they contain a strategically placed crosslink to limit conformational space to more collapsed conformations. The crosslink is designed with the ideal that its primary function is to restrict the mobility (entropy) of the chain, rather than to stabilize folded structure. Core modules are of great interest in themselves, and it is proposed to characterize their conformational ensembles and to optimize their stabilities of their native-like conformation(s). Further, strategies have been developed to link core modules in tandem to construct a larger protein with a true native state. Integrated throughout this research are continued improvements and optimizations of chemistry and purification tools for efficient preparation of homogeneous small protein analogues, including issues involved with the efficient creation of disulfide, thioether, side-chain lactam, and head-to-tail lactam crossbridges. The ongoing and proposed studies exemplify new approaches and are leading to significant and generalizable insights that contribute to the "protein folding problem."