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集合的变化如何调节它们的催化功能。 理解这种关系对于理解大分子现象至关重要， 作为变构调节，但它仍然很困难，因为相关的构象变化 涉及跨越宽长度尺度（亚纳米到多纳米）发生的运动的层次， 时间尺度（ps-s）。我们的实验室正在开发新一代的结构测量， 联合收割机将温度扰动与静态和时间分辨X射线晶体学相结合， 我们探索蛋白质分子的构象景观细节。我们的目标是应用 这些方法用于从关键酶家族，包括激酶， 蛋白酶和ATP依赖性伴侣，以了解它们的构象变化 系综调节它们的生物功能。我们工作的具体目标是：（1）利用多媒体技术， 温度X射线晶体学，结合传统的生物化学和生物物理 测定，以量化构象状态和催化活性之间的关系。（二） 表征以前不可见的酶构象状态，包括隐藏口袋 这可以作为药物发现的目标，使用时间分辨温度跳跃晶体学。 (3)继续开发新的硬件和软件，以改进对 多温度和温度突变晶体学的数据。我们的研究代表了 了解活性和非活性构象平衡如何驱动的新方法 蛋白质功能的调节。成功完成将产生关于 生物学和临床上重要酶的结构-功能关系，并提供新的 给他们提供了治疗的机会。我们预计蛋白质的类似变化 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是普遍有用的 更广泛地理解变构调节。最后，我们的工作将制定一个框架， 研究蛋白质结构、动力学和功能之间的关系， 蛋白质构象集合对温度的反应，我们的目标是民主化 使用多温度和温度跃变晶体学作为一种通用工具， 结构生物学社区

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.