Mapping and rewiring protein allostery

项目摘要。 生物调节依赖于蛋白质变构，其中蛋白质中一个位点的扰动导致蛋白质中一个位点的改变。 远端部位的功能改变。因为变构的表征挑战了我们技术的极限， 能力-需要同时观察的结构，动力学，功能和能量的变化， a蛋白质系综-很少有变构机制被理解为原子细节。这种知识差距限制了 我们对变构是如何在疾病中被保存、进化或影响的基本理解，以及我们的 设计新的变构蛋白的能力。弥合差距需要有方法来映射、验证和调整特定的 这些相互作用驱动蛋白质改变其平衡以占据不同的功能状态。 我是NIH IRACDA和UCSF校长的博士后研究员。我的导师是塔尼娅医生 计算生物物理学专家Kortemme。在过去的三年里，我设计并描述了一种 人工蛋白质生物传感器使用计算蛋白质设计方法和生物物理技术。这项工作 是在蛋白质-蛋白质界面中重新设计小分子结合位点以构建 功能性的模块化传感器/致动器系统。在这份提案中，我的目标是建立一个新的平台， 在变构蛋白中表征和工程化信号转导。在我的共同导师博士的指导下， Susan Marqusee是氢交换方法的先驱，我在目标1中提出了映射变构和能量 使用氢-氘在乳糖阻遏物（LacI）中的蛋白质残基、配体和溶剂之间的偶联 质谱（HX/MS）和等温滴定量热法。在博士的指导下， Kortemme，我将通过重新设计LacI将其变构反应转换为 不同的配体，建立一个策略来控制蛋白质的构象平衡。在目标2中，我建议 将这种方法扩展到糖响应性转录因子的LacI/GalR家族中的蛋白质，以确定 并验证变构在结构和功能相关蛋白质中的保守程度。在Aim中 3.我将应用我们的平台来描述一个古老的，高度进化的 保守蛋白质，并了解癌症相关突变如何影响其机制。在我的支持下 导师，合作者，和更大的研究环境在加州大学旧金山分校和加州大学伯克利分校，我将在HX/MS培训， 计算方法的发展，大型数据集的定量分析，图书馆建设，以及高 通量筛选这些技能将帮助我把我的生化工程背景和我的 迷恋蛋白质调控，建立一个成功的独立研究计划，调查 控制变构的基本原理，以及设计新的治疗性蛋白质。

Project summary. Biological regulation depends on protein allostery, in which a perturbation at one site in a protein causes a functional change at a distal site. Because characterization of allostery challenges the limits of our technical abilities – requiring simultaneous observation of changes in the structure, dynamics, function and energetics of a protein ensemble – few allosteric mechanisms are understood in atomistic detail. This knowledge gap limits our fundamental understanding of how allostery is conserved, evolved, or affected in disease, as well as our abilities to design new allosteric proteins. Bridging the gap requires methods to map, validate and tune specific interactions that drive proteins to shift their equilibria to occupy different functional states. I am an NIH IRACDA and UCSF Chancellor's Postdoctoral fellow at UCSF. My mentor is Dr. Tanja Kortemme, an expert in computational biophysics. In the last three years I engineered and characterized an artificial protein biosensor using computational protein design methods and biophysical techniques. This work is the first example of the de novo design of a small molecule binding site in a protein-protein interface to build a functional, modular sensor/actuator system. In this proposal, I aim to establish a new platform for characterizing and engineering signal transduction in allosteric proteins. With the guidance of my co-mentor Dr. Susan Marqusee, a pioneer in hydrogen exchange methods, I propose in Aim 1 to map allosteric and energetic coupling among protein residues, ligands and solvent in the lac repressor (LacI) using hydrogen-deuterium exchange with mass spectrometry (HX/MS) and isothermal titration calorimetry. With the mentorship of Dr. Kortemme, I will computationally test this mechanism by reengineering LacI to switch its allosteric response to different ligands, establishing a strategy to control conformational equilibria in proteins. In Aim 2, I propose to extend this approach to proteins in the LacI/GalR family of sugar-responsive transcription factors to determine and validate to what extent allostery is conserved among structurally and functionally related proteins. In Aim 3, I will apply our platform to characterize the evolution and regulation of allostery in an ancient, highly conserved protein, and learn how cancer-associated mutations impact its mechanism. With the support of my mentors, collaborators, and the greater research environment at UCSF and UC Berkeley, I will train in HX/MS, computational methods development, quantitative analysis of large datasets, library construction, and high- throughput screening. These skills will help me bridge my background in biochemical engineering with my fascination with protein regulation to build a successful independent research program investigating the fundamentals principles that govern allostery, and engineering new therapeutic proteins.