Exploiting temperature-sensitive orthologs to understand protein allostery

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。 通过研究它们的构象系综的变化如何调节它们的催化功能。 理解这种关系对于理解这样的大分子现象至关重要 作为变构调节，然而它仍然很困难，因为相关的构象变化 涉及跨宽长度尺度(亚欧到多纳米)发生的运动的层次结构 时间尺度(PS-S)。我们的实验室正在开发新一代结构测量方法， 将温度扰动与静态和时间分辨X射线结晶学相结合，允许 我们将详细探索蛋白质分子的构象图景。我们的目标是申请 这些方法针对来自关键酶家族的温度敏感的同源基因，包括激酶， 蛋白水解酶和ATP依赖的伴侣蛋白，以了解其构象如何变化 合奏调节它们的生物功能。我们工作的具体目标是：(1)使用多个 温度X射线结晶学，结合传统的生化和生物物理 实验，量化构象状态和催化活性之间的关系。(2) 表征以前看不见的酶的构象状态，包括隐蔽的口袋 使用时间分辨温度跳跃结晶学，这可以作为药物发现的目标。 (3)继续开发新的硬件和软件，以改进数据的收集和分析 来自多温度和温度跳跃的结晶学数据。我们的研究代表了一种 理解活性构象和非活性构象平衡如何驱动的新方法 蛋白质功能的调节。成功完成后，将生成有关 生物和临床重要酶的结构-功能关系并提供新的 以治疗为靶点的机会。我们预计蛋白质也会发生类似的变化 构象系综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对 更广泛地理解变构调节。最后，我们的工作将制定一个框架，以 研究蛋白质结构、动力学和功能之间的关系 蛋白质构象系综对温度的响应，我们的目标是使 使用多温度和温度跳跃结晶学作为一种通用工具 结构生物界。

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.