Folding of Dihydrofolate Reductase and the Response Regulators

二氢叶酸还原酶的折叠和响应调节剂

• 批准号: 1121942
• 负责人: C Robert Matthews
• 金额: $ 69.99万
• 依托单位: University of Massachusetts Medical School
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-08-01 至 2014-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1121942&HistoricalAwards=false
• 关键词: Folding; Dihydrofolate; Reductase; Response; Regulators

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.

该项目的目的是检验以下假设：在非常常见的Rossmann折叠蛋白家族的两个成员Chey和Dihyrofaly还原酶（DHFR）的两个成员中，链连通性与疏水簇之间的相互作用决定了它们的折叠自由能表面。两种蛋白质的可用证据都表明，异亮氨酸，亮氨酸和缬氨酸侧链的局部连接簇通过子域迅速塌陷，这些子域可以增强或阻碍随后导致天然构象的随后折叠反应。 在均衡状态下，以平衡和连接到超高式混合系统的平衡和连接的光谱方法将探测化学贬低状态的大小，形状和成对距离，并以部分折叠的状态探测出在微秒时间内出现在稀释到Chey的本地培养条件后的微秒时间范围内的，以补充DHFR的先前发现。 化学移位指数和顺磁性松弛增强NMR测量将探测化学变性状态下的非随机结构。 Chey上的互补脉冲液体交换实验将探测肽在早期中间体中二级结构的形成和位点特异性水平。 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在推动这些簇以及N-和C-末端亚域的形成中连通性的作用。 适当的Chey的置换变体将进行单分子拉力实验，以研究亚域连接性和ILV群集完整性对展开反应的合作性的影响，并揭示部分折叠状态的稳定。 实验数据将用于验证Chey和DHFR的折叠反应的课程元素MD模拟以及Chey上的高分辨率模拟。 可以预料，实验和计算方法在同一目标上的合并应用将显着提高方法的价值并加快蛋白质折叠问题的解决方案。蛋白质折叠问题仍然是分子生物物理学的重要挑战之一，其解决方案将对生物学和生物技术产业产生重大影响。 为了加快解决折叠问题的解决方案，已经建立了研究人员的协作网络，以在单个蛋白质靶标上生成全面的实验数据，该数据将验证伴随的粗粒和高分辨率MD折叠反应的MD模拟。 这种协作方法将成为解决生物学其他复杂问题的范式。 在这项工作的过程中，已经开发了一个微通道混合系统，该系统允许访问微秒折叠反应，并且可以连接到各种光谱方法。 这项技术已与其他机构的同事共享，其在开放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。 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，并且科学进步已纳入研究生分子生物物理学课程中。