Miniproteins: Folding Equilibria, Pathways and Rates

微蛋白：折叠平衡、途径和速率

• 批准号: 6918148
• 负责人: Niels Hjorth Andersen
• 金额: $ 27.22万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6918148
• 关键词: chemical kinetics; chemical substitution; computational biology; computer simulation; gene mutation; intermolecular interaction; model design /development; molecular dynamics; nuclear magnetic resonance spectroscopy; physical model; protein folding; protein structure function; structural biology

DESCRIPTION (provided by applicant): The efficient folding of polypeptide sequences into stable structures is one of the most fascinating and fundamentally important biorecognition phenomena. Studies of fold stability as well as folding pathways and rates are often more incisive when performed on minimalist constructs rather than full-sized proteins. The designed Trp-cage fold, consisting of only 18-19 essential residues, has proven to be an excellent system for measuring the fold stabilization contributions of intrinsic secondary structure preferences and individual hydrophobic, coulombic (saltbridge), and H-bonding interactions. Additional mutational studies of the Trp-cage are proposed. These, in conjunction with molecular dynamics simulations of folding trajectories, are aimed at determining, in detail, the pathway(s) of Trp-cage folding and at discovering alternate packing arrangements that could serve as the hydrophobic cores for related miniprotein motifs. Folding rates and cooperativity will be determined by several techniques (NMR line broadening, NMR chemical shift melts, and fluorescence-monitored T-jumps) to ascertain if the rates are dependent on contact order at the lower range of protein size. Extensively-mutated Trp-cage constructs, truncations and circular permutants will figure heavily in these studies. The present proposal continues to address polypeptide structuring requisites by both de novo design and as a fold optimization problem, but with an increasing emphasis on folding pathways and rates. The folding rate determinations should define the source (increased folding rate or decreased unfolding rate) of each of the structure-stabilizing interactions in the Trp-cage. Mutational effects on the rates of b-hairpin and three-stranded sheet formation will also be determined using dynamic NMR methods. Binding and novel structure stabilizing effects of the Trp side chain will be explored and quantitated. The design, construction, and optimization of a BaB3 miniprotein (a parallel b sheet resulting from the association of b strands at each end of an a helix) are also proposed. This construct mimics some features of the Bl domain of protein L. The designed BaB target fold is, however, novel in two major respects: the helix runs in the opposite direction of that in the Bl domain, and there is a left-handed crossover from a B strand to an alpha helix - the latter has never been observed in nature. The studies outlined in this proposal will provide structural and mechanistic insights concerning the requisites for fast and efficient folding that should yield protein engineering strategies for reducing disease-related misfolding events. More accurate methods for quantitating polypeptide structuring and folding rates will also result.

描述（由申请人提供）：将多肽序列有效地折叠成稳定的结构是最引人入胜且根本重要的生物识别现象之一。折叠稳定性以及折叠途径和速率的研究通常在极简主义构建体而不是全尺寸蛋白质上进行。事实证明，设计的TRP笼折叠仅由18-19个基本残基组成，是一个极好的系统，用于测量固有二级结构偏好和单个疏水性，库仑（Saltbridge）和H键相互作用的折叠稳定贡献。提出了TRP笼的其他突变研究。这些结合了折叠轨迹的分子动力学模拟，旨在详细确定TRP-Cage折叠的途径，并发现可以用作相关微蛋白基序的疏水核心的替代填料布置。折叠率和协作性将由多种技术（NMR线扩展，NMR化学移位熔体和荧光监测的T-跳跃）确定，以确定速率是否取决于蛋白质大小较低范围的接触顺序。在这些研究中，广泛突出的TRP式构建体，截断和圆形排列物将很大程度上构想。本提案继续通过从头设计和折叠优化问题来解决多肽结构的要求，但越来越强调折叠途径和速率。折叠速率的确定应定义TRP笼中每种结构稳定相互作用的源（增加折叠率或下降速率）。也将使用动态NMR方法确定对B发宾和三链薄板形成速率的突变作用。 TRP侧链的结合和新结构稳定效应将被探索和定量。还提出了BAB3小蛋白的设计，构造和优化（还提出了A螺旋的每一端的B链相关的平行B片）。该构建体模拟了蛋白质L的BL结构域的某些特征。但是，设计的BAB目标折叠在两个主要方面是新颖的：螺旋在BL域中的相反方向延伸，并且从B链到Alpha Helix的左手交叉 - 后者在本质上从未观察到后者。该提案中概述的研究将提供有关快速有效折叠必需品的结构和机械见解，这些折叠应产生蛋白质工程策略，以减少与疾病相关的错误折叠事件。定量多肽结构和折叠率的更准确的方法也将导致。