Folding Mechanisms of Dihydrofolate Reductase and the Response Regulators

二氢叶酸还原酶的折叠机制及其响应调节剂

• 批准号: 0721312
• 负责人: C Robert Matthews
• 金额: $ 57万
• 依托单位: University of Massachusetts Medical School
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-07-15 至 2011-06-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0721312&HistoricalAwards=false
• 关键词: Folding; Mechanisms; Dihydrofolate; Reductase; Response

The goal of this project is to perform a comparative analysis of the folding mechanisms of two members of the alpha/beta/alpha sandwich motif, one of the most common in biology. Previous NSF-supported studies of the mechanism of folding of one sub-class of this motif, dihydrofolate reductase (DHFR), have shown that this small two-domain protein folds via a sequential set of on-pathway partially-folded states in four parallel channels. By contrast, two members of the flavodoxin fold family, another sub-class of this motif, initially misfold to an off-pathway intermediate that must at least partially unfold to access the productive transition state leading to the native conformation. The molecular basis for this misfolding reaction will be probed by a combination of experimental and computational methods that will focus on determining the structural features and energetics of these intermediates for CheY, NtrC and Spo0F, all members of the response regulator sub-class of the flavodoxin fold. Continuous-flow (CF) small-angle x-ray scattering (SAXS), time-resolved Forster resonance energy transfer (trFRET) and far-UV circular dichroism (CD) measurements of microsecond folding reactions will provide quantitative information on dimensional properties and global secondary structure. Pulse-quench hydrogen exchange methodology and mass spectrometric analysis of mass-labeled peptides will enhance the circular dichroism data by identifying the segments that define the cores of stability. Mutational analysis will explore the roles of individual side chains in the folding and misfolding of CheY, with a particular focus on large nonpolar side chains in the pair of hydrophobic cores on either side of the central beta-sheet. The role of chain entropy in the CheY misfolding reaction will be tested by creating permutations that vary the chain connectivity while preserving 3D structure. Collaborative native-centric simulations of response regulator folding mechanisms will provide detailed structural insights into the misfolded species and the process by which they backtrack to the native conformations. Comparisons with the results for the on-pathway sub-millisecond folding reaction in DHFR will provide additional insights into the contrast with response regulators. The results of this multi-dimensional comparative analysis are expected to enhance the understanding of early folding reactions in alpha/beta/alpha sandwich proteins and the basis for the misfolding reactions in the flavodoxin fold.The broader impact of this project has training, educational and technology development components. Undergraduate, graduate and postdoctoral fellows will receive training in molecular biophysics, including spectroscopic methods, thermodynamics and kinetics, sophisticated data analysis, protein engineering, and the protein folding problem. Several of the methods employed, including circular dichroism and fluorescence spectroscopy, and data analysis algorithms developed will form a part of a new advanced topics course in Biophysical Methods for graduate students in the PI's Department. The analytical tools developed during the course of this research on protein folding mechanisms are currently being used in over a dozen research labs around the world, and will be made more accessible via a website sponsored by the Biochemistry and Molecular Pharmacology Department. The continuous-flow mixing technology developed during the previous grant period will continue to be optimized and available to all users by collaboration (for CF-trFRET and CF-CD) or through the BioCAT beamline at the Advanced Photon Source at Argonne National Laboratory (for CF-SAXS).

该项目的目标是对 α/β/α 三明治图案（生物学中最常见的图案之一）的两个成员的折叠机制进行比较分析。 先前 NSF 支持的对该基序的一个亚类二氢叶酸还原酶 (DHFR) 折叠机制的研究表明，这种小型双结构域蛋白通过一组连续的路径上部分折叠状态以四个平行的方式折叠。渠道。 相比之下，黄素氧还蛋白折叠家族（该基序的另一个子类）的两个成员最初错误折叠成非途径中间体，该中间体必须至少部分展开才能进入产生天然构象的生产性过渡状态。 这种错误折叠反应的分子基础将通过实验和计算方法相结合来探索，重点是确定 CheY、NtrC 和 Spo0F 这些中间体的结构特征和能量学，这些中间体都是黄素氧还蛋白的反应调节子类的成员折叠。 微秒折叠反应的连续流 (CF) 小角 X 射线散射 (SAXS)、时间分辨福斯特共振能量转移 (trFRET) 和远紫外圆二色性 (CD) 测量将提供有关尺寸特性和全局的定量信息二级结构。 脉冲淬灭氢交换方法和质量标记肽的质谱分析将通过识别定义稳定性核心的片段来增强圆二色性数据。 突变分析将探讨各个侧链在 CheY 折叠和错误折叠中的作用，特别关注中央 β 折叠片两侧一对疏水核心中的大非极性侧链。 将通过创建改变链连接性同时保留 3D 结构的排列来测试链熵在 CheY 错误折叠反应中的作用。 对反应调节器折叠机制的以原生为中心的协作模拟将为错误折叠物种及其回溯到原生构象的过程提供详细的结构见解。 与 DHFR 中路径上亚毫秒折叠反应的结果进行比较，将为与反应调节剂的对比提供更多见解。 这一多维比较分析的结果预计将增强对 α/β/α 夹心蛋白中早期折叠反应的理解，以及黄素氧还蛋白折叠中错误折叠反应的基础。该项目的更广泛影响包括培训、教育和技术开发组件。 本科生、研究生和博士后研究员将接受分子生物物理学方面的培训，包括光谱方法、热力学和动力学、复杂的数据分析、蛋白质工程和蛋白质折叠问题。 所采用的几种方法，包括圆二色性和荧光光谱法，以及开发的数据分析算法，将成为 PI 系研究生的生物物理方法新高级主题课程的一部分。 在这项蛋白质折叠机制研究过程中开发的分析工具目前正在世界各地的十几个研究实验室使用，并将通过生物化学和分子药理学系赞助的网站更容易访问。 上一资助期间开发的连续流混合技术将继续优化，并通过合作（用于 CF-trFRET 和 CF-CD）或通过阿贡国家实验室先进光子源的 BioCAT 光束线（用于CF-SAXS）。