Miniproteins: Folding Equilibria, Pathways and Rates

微蛋白：折叠平衡、途径和速率

• 批准号: 7038338
• 负责人: Niels Hjorth Andersen
• 金额: $ 26.75万
• 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/7038338
• 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）：将多肽序列有效折叠成稳定结构是最令人着迷且最重要的生物识别现象之一。在极简结构上进行的折叠稳定性以及折叠途径和速率的研究通常比在全尺寸蛋白质上进行的研究更加深入。设计的色氨酸笼折叠仅由 18-19 个必需残基组成，已被证明是一个出色的系统，可用于测量固有二级结构偏好和单个疏水性、库仑（盐桥）和氢键相互作用的折叠稳定性贡献。提出了色氨酸笼的额外突变研究。这些与折叠轨迹的分子动力学模拟相结合，旨在详细确定色氨酸笼折叠的途径，并发现可作为相关小蛋白基序的疏水核心的替代包装排列。折叠速率和协同性将通过多种技术（NMR 谱线展宽、NMR 化学位移熔解和荧光监测 T 跃迁）来确定，以确定折叠速率是否取决于蛋白质尺寸较低范围内的接触顺序。广泛突变的色氨酸笼结构、截短和环状排列将在这些研究中占据重要地位。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。折叠速率测定应定义色氨酸笼中每个结构稳定相互作用的来源（增加的折叠速率或降低的展开速率）。突变对 b-发夹和三链片形成速率的影响也将使用动态 NMR 方法来确定。将探索和定量色氨酸侧链的结合和新颖结构稳定作用。还提出了 BaB3 微型蛋白（由 a 螺旋两端的 b 链结合产生的平行 b 片层）的设计、构建和优化。该构建体模仿了蛋白质 L 的 Bl 结构域的一些特征。然而，设计的 BaB 靶折叠在两个主要方面是新颖的：螺旋以与 Bl 结构域中的方向相反的方向运行，并且存在左手交叉从B链到α螺旋——后者在自然界中从未被观察到。该提案中概述的研究将提供有关快速有效折叠的必要条件的结构和机制见解，从而产生减少与疾病相关的错误折叠事件的蛋白质工程策略。还将产生更准确的定量多肽结构和折叠率的方法。