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.

这个项目的目标是测试的假设，链连接和疏水簇的支链脂肪族侧链之间的相互作用，在两个成员的非常常见的罗斯曼折叠蛋白质家族，CheY和二氢叶酸还原酶（DHFR），决定他们的折叠自由能表面。关于这两种蛋白质的现有证据表明，局部连接的异亮氨酸、亮氨酸和缬氨酸侧链簇通过亚结构域快速塌陷，亚结构域可以增强或阻碍随后的折叠反应，导致天然构象。 一组光谱方法，在平衡和接口的超快速混合系统，将探测的大小，形状和成对的距离在化学变性状态和部分折叠状态，出现在微秒的时间范围内稀释后的本地有利于条件的CheY，补充以前的研究结果DHFR。 化学位移指数和顺磁弛豫增强NMR测量将探测化学变性状态下的非随机结构。 对CheY的互补脉冲猝灭氢交换实验将探测肽二级结构的形成和早期中间体的位点特异性水平。 突变分析将测试本地和非本地ILV簇在驱动这些早期折叠反应中的作用，并且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-末端亚结构域的形成中的作用。 适当的排列变体的CheY将进行单分子拉动实验，以研究子域连接性和ILV簇完整性对展开反应的协同性的影响，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折叠反应的粗粒度MD模拟以及CheY的高分辨率模拟。 蛋白质折叠问题是分子生物物理学中的一个突出难题，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，已经建立了一个研究人员的协作网络，以生成一个关于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，已经开发了一个微通道混合系统，允许进入微秒折叠反应，并可以连接到各种光谱方法。 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。 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，目前正在将科学进展纳入研究生分子生物物理学课程。