Exploiting temperature-sensitive orthologs to understand protein allostery

利用温度敏感的直系同源物来了解蛋白质变构

• 批准号: 10716051
• 负责人: Michael C. Thompson
• 金额: $ 37.49万
• 依托单位: UNIVERSITY OF CALIFORNIA, MERCED
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2028-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10716051
• 关键词: Allosteric Regulation; Biochemical; Biological Assay; Biological Phenomena; Biological Process; Biophysics; Clinical; Collection; Communities; Computer software; Crystallography; Data Analyses; Democracy; Disease; Drug Targeting; Enzymes; Equilibrium; Family; Generations; Goals; Health; Human Biology; Knowledge; Length; Measurement; Methods; Molecular; Molecular Chaperones; Molecular Conformation; Motion; Orthologous Gene; Peptide Hydrolases; Phosphotransferases; Protein Conformation; Proteins; Regulation; Research; Structure; Structure-Activity Relationship; Temperature; Therapeutic; Time; Work; X-Ray Crystallography; biophysical properties; drug discovery; improved; insight; novel strategies; protein function; protein structure; response; structural biology; temperature jump; temporal measurement; time use; tool

Project Summary We propose to study the relationship between the structure, dynamics, and function of enzymes by examining how changes to their conformational ensembles regulate their catalytic functions. Understanding this relationship is critical for understanding macromolecular phenomena such as allosteric regulation, yet it remains difficult, because the relevant conformational changes involve a hierarchy of motions that occur across broad lengthscales (sub-Å to multi-nm) and timescales (ps-s). Our lab is developing a new generation of structural measurements that combine temperature perturbations with static and time-resolved X-ray crystallography, allowing us to explore the conformational landscapes of protein molecules in detail. We aim to apply these methods to temperature-sensitive orthologs from key enzyme families, including kinases, proteases, and ATP-dependent chaperones, to understand how changes to their conformational ensembles modulate their biological functions. The specific goals of our work are: (1) Use multi- temperature X-ray crystallography, combined with traditional biochemical and biophysical assays, to quantify the relationship between conformational states and catalytic activity. (2) Characterize previously invisible conformational states of enzymes, including cryptic pockets that can be targeted for drug discovery, using time-resolved temperature-jump crystallography. (3) Continue developing new hardware and software to improve the collection and analysis of data from multi-temperature and temperature-jump crystallography. Our research represents a novel approach to understanding how the balance of active and inactive conformations drives the regulation of protein function. Successful completion will yield new information about the structure-function relationships of biologically and clinically important enzymes and provide new opportunities for targeting them with therapeutics. We expect that similar changes to protein conformational ensembles underlie thermal regulation and other types of allosteric regulation in these enzyme families, and therefore we expect our results to be generally useful in understanding allosteric regulation more broadly. Finally, our work will develop a framework for studying the relationship between protein structure, dynamics, and function that exploits the response of protein conformational ensembles to temperature, and we aim to democratize the use of multi-temperature and temperature-jump crystallography as a general tool for the structural biology community.

项目摘要 我们建议研究酶的结构，动力学和功能之间的关系 通过检查如何改变其构象合奏，可以调节其催化功能。 理解这种关系对于理解大分子现象至关重要 作为变构法规，但仍然很困难，因为相关构象变化 涉及跨长度尺度（sub-Å至多NM）和 时间尺度（PS-S）。我们的实验室正在开发新一代的结构测量结果 将温度扰动与静态和时间分辨的X射线晶体学结合在一起，允许 美国详细探讨蛋白质分子的构象景观。我们的目标是申请 这些方法来自关键酶家族的温度敏感直系同源物，包括激酶， 蛋白酶和依赖ATP的伴侣，以了解其构象如何变化 合奏调节其生物学功能。我们工作的具体目标是：（1）使用多 温度X射线晶体学，结合传统的生化和生物物理 测定，以量化构象状态与催化活性之间的关系。 （2） 表征先前看不见的酶的构象状态，包括加密口袋 可以使用时间分辨的温度悬浮晶体学来针对药物发现。 （3）继续开发新的硬件和软件，以改善对的收集和分析 来自多温度和温度跳跃晶体学的数据。我们的研究代表 了解主动和不活动构象如何驱动的新方法 蛋白质功能的调节。成功完成将产生有关 生物学和临床上重要的酶的结构功能关系，并提供新的 针对治疗的机会。我们期望蛋白质类似的变化 构象合奏是热调节和其他类型的变构调节的基础 这些酶家族，因此我们希望我们的结果通常在 更广泛地了解变构调节。最后，我们的工作将为 研究利用蛋白质结构，动力学和功能之间的关系 蛋白质构象合奏对温度的反应，我们旨在使人民主化 使用多温度和温度悬浮晶体学作为一般工具 结构生物学社区。