Folding of Dihydrofolate Reductase and the Response Regulators

这个项目的目的是测试一个假设，即链连性和支链脂肪侧链的疏水簇之间的相互作用，在两个非常常见的罗斯曼折叠蛋白家族的成员，CheY和二氢叶酸还原酶（DHFR），决定了它们的折叠自由能表面。现有证据表明，这两种蛋白质的异亮氨酸、亮氨酸和缬氨酸侧链的局部连接簇通过亚结构域迅速坍塌，这些亚结构域可以增强或阻碍随后的折叠反应，从而导致天然构象。在平衡态和超快速混合系统的界面上，一系列光谱方法将探测化学变性态和部分折叠态的大小、形状和成对距离，这些状态在稀释到有利于CheY的天然条件后出现在微秒时间范围内，补充先前在DHFR上的发现。化学位移指数和顺磁弛豫增强核磁共振测量将探测化学变性状态下的非随机结构。CheY的互补脉冲猝灭氢交换实验将探索肽二级结构的形成和早期中间体的位点特异性水平。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通性在驱动这些簇和N端和c端亚结构域形成中的作用。将对CheY的适当排列变体进行单分子拉实验，以研究子结构域连通性和ILV簇完整性对展开反应协同性的影响，并揭示部分折叠态的稳定性。实验数据将用于验证CheY和DHFR折叠反应的粗粒度MD模拟，以及CheY的高分辨率模拟。预计实验和计算方法在同一目标上的结合应用将大大提高两种方法的价值，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一个突出的挑战，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，研究人员建立了一个合作网络，以生成单个蛋白质靶点的综合实验数据集，以验证其折叠反应的粗粒度和高分辨率MD模拟。这种合作的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，一种微通道混合系统已经被开发出来，它可以实现微秒级的折叠反应，并且可以与各种光谱方法相结合。这项技术已与其他机构的同事分享，其在公开文献中的传播使其他人能够研究其目标系统中的早期折叠事件。对这些研究目标的追求也将为高中生、本科生、研究生和博士后提供培训机会，并且科学进展正在被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.