Miniproteins: Folding Equilibria, Pathways and Rates

描述(申请人提供)：多肽序列有效地折叠成稳定的结构是最吸引人和最重要的生物识别现象之一。对折叠稳定性以及折叠路径和速率的研究通常在最低限度的结构上进行，而不是在全尺寸蛋白质上进行。设计的Trp-笼状折叠只由18-19个基本残基组成，已被证明是一个很好的系统，可以测量固有的二级结构偏好以及单个疏水、库仑(盐桥)和氢键相互作用对折叠稳定的贡献。建议对Trp-Cage进行更多的突变研究。这些结合折叠轨迹的分子动力学模拟，旨在详细确定Trp-笼状折叠的途径(S)，并发现可作为相关小蛋白基序疏水核心的替代堆积排列。折叠率和协同性将通过几种技术(核磁共振谱线加宽、核磁共振化学位移熔化和荧光监测的T-跳跃)来确定在较低的蛋白质尺寸范围内，折叠率是否取决于接触顺序。广泛突变的Trp-笼状结构、截断和环形置换将在这些研究中发挥重要作用。本提案继续通过从头设计和作为折叠优化问题来解决多肽结构的必要条件，但越来越强调折叠路径和速率。折叠速度的测定应该确定Trp-笼子中每个稳定结构的相互作用的来源(增加的折叠速度或减少的展开速度)。突变对b-发夹和三链片层形成速率的影响也将用动态核磁共振方法来确定。将探索和量化色氨酸侧链的结合和新的结构稳定作用。还提出了BaB3微型蛋白(由a螺旋两端的b链结合而成的平行b片层)的设计、构建和优化。然而，设计的Bab靶向折叠在两个主要方面是新颖的：螺旋与Bl结构域中的螺旋方向相反，以及从B链到阿尔法螺旋的左手交叉-后者在自然界中从未被观察到。本提案中概述的研究将提供关于快速和有效折叠的必要条件的结构和机械见解，这将产生减少与疾病相关的错误折叠事件的蛋白质工程策略。也将产生更准确的多肽结构和折叠率的定量方法。

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.