Miniproteins: Folding Equilibria, Pathways and Rates

描述（由申请人提供）：多肽序列有效折叠成稳定结构是最迷人和最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结构上进行，而不是在全尺寸蛋白质上进行。所设计的Trp-cage折叠仅由18-19个基本残基组成，已被证明是测量固有二级结构偏好和单个疏水、库仑（盐桥）和氢键相互作用的折叠稳定性贡献的优秀系统。提出了对trp笼的其他突变研究。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型折叠的途径，并发现可作为相关微小蛋白基序疏水核心的替代包装安排。折叠速率和协同性将通过几种技术（核磁共振谱线展宽、核磁共振化学位移熔体和荧光监测的t跳）来确定，以确定速率是否依赖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、截断和环状置换将在这些研究中占据重要地位。目前的建议继续通过从头设计和折叠优化问题来解决多肽结构要求，但越来越强调折叠途径和速率。折叠速率的测定应该确定trp笼中每个结构稳定相互作用的来源（增加折叠速率或减少展开速率）。突变对b发夹和三链薄片形成速率的影响也将使用动态核磁共振方法确定。将探索和量化Trp侧链的结合和新型结构稳定作用。本文还提出了BaB3微型蛋白（由a螺旋两端的b链结合而成的平行b片）的设计、构建和优化。这种结构模仿了蛋白质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：螺旋与Bl结构域的方向相反，并且从B链到α螺旋存在左手交叉-后者在自然界中从未观察到过。本提案中概述的研究将为快速有效折叠的必要条件提供结构和机制方面的见解，从而产生减少疾病相关错误折叠事件的蛋白质工程策略。更准确的定量多肽结构和折叠率的方法也将产生。

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.