Folding of Dihydrofolate Reductase and the Response Regulators

本项目的目标是检验这一假设，即在非常常见的Rossmann-Fold家族的两个成员-Chey和二氢叶酸还原酶(DHFR)中，链连接性和支链脂肪族侧链的疏水簇之间的相互作用决定了它们的折叠自由能表面。对这两种蛋白质的现有证据表明，异亮氨酸、亮氨酸和缬氨酸侧链的局部连接簇通过亚域迅速崩溃，这些亚域可以加强或阻碍随后的折叠反应，从而导致天然构象。一组光谱方法，在平衡和接口到超快速混合系统，将探测在化学变性状态和部分折叠状态下的大小、形状和成对距离，这些状态在稀释到对CHEY有利的条件后出现在微秒时间范围内，补充了之前关于DHFR的发现。化学位移指数和顺磁弛豫增强核磁共振测量将探索化学变性状态下的非随机结构。在CHEY上的互补脉冲猝灭氢交换实验将探索在多肽和早期中间体的位置特异性水平上的二级结构的形成。突变分析将测试局部和非局部ILV簇在驱动这些早期折叠反应中的作用，序列的排列将测试多肽链的连接性在驱动这些簇以及N-和C-末端亚域形成中的作用。适当的CHEY置换变体将受到单分子拉动实验，以研究亚区连接性和ILV簇完整性对去折叠反应协同性的影响，并揭示部分折叠状态的稳定性。实验数据将用于验证Chey和DHFR折叠反应的过程粒度MD模拟，以及对Chey的高分辨率模拟。预计在同一目标上结合实验和计算方法将大大提高这两种方法的价值，并加快蛋白质折叠问题的解决。蛋白质折叠问题仍然是分子生物物理学中的突出挑战之一，它的解决将对生物学和生物技术产业产生重大影响。为了加快折叠问题的解决，已经建立了一个合作的调查人员网络，以生成单个蛋白质目标的综合实验数据集，该数据集将验证其折叠反应的伴随粗粒度和高分辨率MD模拟。这种协作方法将成为解决生物学中其他复杂问题的范例。在这项工作的过程中，已经开发了一种微通道混合系统，它允许进入微秒级的折叠反应，并可以与各种光谱方法接口。这项技术已经与其他机构的同事分享，它在开放文献中的传播使其他人能够研究他们目标系统中的早期折叠事件。追求这些研究目标还将为高中生、本科生、研究生和博士后研究员提供培训机会，科学进步正在纳入研究生分子生物物理学课程。

The goal of this project is to test the hypothesis that the interplay between chain connectivity and hydrophobic clusters of branched aliphatic side chains in two members of the very common Rossmann-fold family of proteins, CheY and dihydrofolate reductase (DHFR), dictates their folding free energy surfaces. Available evidence on both proteins suggests that locally-connected clusters of isoleucine, leucine and valine side chains rapidly collapse via subdomains that can enhance or impede subsequent folding reactions leading to the native conformation. A battery of spectroscopic methods, at equilibrium and interfaced to ultra-rapid mixing systems, will probe the size, shape and pair-wise distances in the chemically-denatured state and in partially-folded states that appear in the microsecond time range after dilution to native-favoring conditions for CheY, complementing previous findings on DHFR. Chemical shift index and paramagnetic relaxation enhancement NMR measurements will probe for nonrandom structure in the chemically denatured state. Complementary pulse-quench hydrogen exchange experiments on CheY will probe the formation of secondary structure at the peptide and the site-specific level in the early intermediates. Mutational analysis will test the role of local and nonlocal ILV clusters in driving these early folding reactions, and permutations of the sequences will test the role of the connectivity of the polypeptide chain in driving the formation of these clusters and the N- and C-terminal subdomains. Appropriate permuted variants of CheY will be subjected to single molecule pulling experiments to study the effect of subdomain connectivity and the ILV cluster integrity on the cooperativity of the unfolding reaction and reveal the stabilization of partially-folded states. The experimental data will be used to validate course-grained MD simulations of the folding reactions of CheY and DHFR, and high-resolution simulations on CheY. It is anticipated that the combined application of experimental and computational methods on the same target will substantially enhance the value of both approaches and expedite the solution of the protein folding problem.The protein folding problem remains as one of the outstanding challenges in molecular biophysics, and its solution would have a major impact on biology and the biotechnology industry. To expedite a solution to the folding problem, a collaborative network of investigators has been established to generate a comprehensive experimental data set on a single protein target that will validate companion coarse-grained and high-resolution MD simulations of its folding reaction. This collaborative approach will serve as a paradigm for the solution of other complex problems in biology. A micro-channel mixing system has been developed over the course of this work that allows access to microsecond folding reactions and that can be interfaced to a variety of spectroscopic methods. This technology has been shared with colleagues at other institutions, and its dissemination in the open literature has enabled others to study the early folding events in their target systems. Pursuit of these research objectives will also provide training opportunities for high school students, undergraduates, graduate students and postdoctoral fellows, and the scientific advances are being incorporated into a graduate molecular biophysics course.