SYNTHETIC STUDIES OF PROTEIN STABILITY AND FOLDING

蛋白质稳定性和折叠的综合研究

• 批准号: 6180579
• 负责人: George Barany
• 金额: $ 20.98万
• 依托单位: UNIVERSITY OF MINNESOTA
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 2002-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6180579
• 关键词: bacterial proteins; biophysics; chemical stability; circular dichroism; conformation; crosslink; cyclization; disulfide bond; globular protein; nuclear magnetic resonance spectroscopy; peptide chemical synthesis; protein folding; protein structure function; synthetic peptide; technology /technique development; thermodynamics; trypsin inhibitors

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."

该合作研究项目将最先进的肽和小蛋白质合成的温和化学方法与生物化学和生物物理学研究相结合，以解决基本问题并提出有关蛋白质折叠，稳定性和动力学的假设。亲本小球状蛋白靶点为牛胰腺胰蛋白酶抑制剂（BPTI）， 58个残基与3个二硫化物桥阵列，以及链球菌蛋白G （GB1）的免疫球蛋白结合域，56个不含二硫化物的残基。在支持的第一阶段，研究表明，配对的α -氨基-正丁酸（Abu）同位异构体取代了BPTI中的胱氨酸交联，提供了在温和条件下的蛋白质，其结构与蛋白质折叠过程的前10-20毫秒形成的瞬态中间体非常相似。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，并且它们的元素可以在合适的肽中组合以构建天然样模块。设计的肽长度约为15-50个氨基酸残基，由天然或设计序列连接的核心元件（来自BPTI和/或GB1）组成，它们包含一个战略性的交联，以限制构象空间为更多的折叠构象。交联设计的理想是，其主要功能是限制链的迁移率（熵），而不是稳定折叠结构。核心模块本身非常有趣，并提出了表征它们的构象集成和优化它们的原生构象的稳定性。此外，已经开发出将核心模块串联起来以构建具有真正天然状态的更大蛋白质的策略。在整个研究过程中，不断改进和优化化学和纯化工具，以有效地制备均匀的小蛋白质类似物，包括有效地产生二硫、硫醚、侧链内酰胺和头尾内酰胺交叉桥的问题。正在进行的和提议的研究举例说明了新的方法，并导致了对“蛋白质折叠问题”的重要和可推广的见解。