Mapping and rewiring protein allostery

蛋白质变构的映射和重新布线

• 批准号: 10609154
• 负责人: Anum Azam Glasgow
• 金额: $ 24.9万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10609154
• 关键词: 6-Phosphofructokinase; ART protein; Affect; Allosteric Regulation; Allosteric Site; Behavior; Binding; Binding Sites; Biochemical; Biological; Biophysics; Biosensor; Buffers; Calorimetry; Complex; Computing Methodologies; Consumption; Coupling; DNA; DNA Binding; Data; Deuterium; Disease; Distal; Drug Design; Engineering; Environment; Enzymes; Equilibrium; Escherichia coli; Eukaryota; Evolution; Exhibits; Family; Feedback; Foundations; Gatekeeping; Gene Expression; Gene Library; Glycolysis; Goals; Homologous Protein; Human; Hydrogen; Hydrogen Bonding; Individual; Interferometry; Knowledge; Laboratories; Lac Repressors; Learning; Libraries; Ligand Binding; Ligands; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Measurement; Mediating; Mentors; Mentorship; Methods; Molecular Conformation; Motion; Movement; Mutagenesis; Mutation; Peptides; Pharmaceutical Preparations; Phase; Postdoctoral Fellow; Protein Conformation; Protein Engineering; Protein Family; Protein Isoforms; Proteins; Regulation; Research; Signal Transduction; Site; Solvents; Specificity; Structure; System; Techniques; Testing; Therapeutic; Time; Titrations; Toxic effect; Training; United States National Institutes of Health; Water; Work; base; biophysical properties; biophysical techniques; design; disease diagnosis; experimental study; fascinate; high throughput screening; human disease; improved; innovation; large datasets; member; method development; mutant; mutation screening; novel strategies; novel therapeutics; programs; repaired; response; sensor; simulation; skills; small molecule; sugar; therapeutic protein; transcription factor

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.

项目总结。 生物调节依赖于蛋白质变构，在这种情况下，蛋白质中一个位置的扰动会导致 远端部位的功能改变。因为变构的表征挑战了我们的技术极限 能力--需要同时观察大脑结构、动力学、功能和能量学的变化 蛋白质系综-很少有变构机制被原子细节所理解。这一知识差距限制了 我们对变构如何在疾病中被保守、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。弥合差距需要方法来绘制、验证和调整特定的 驱动蛋白质改变其平衡以占据不同功能状态的相互作用。 我是NIH IRACDA的一名成员，也是加州大学旧金山分校校长的博士后研究员。我的导师是坦贾医生 计算生物物理学专家Kortemme说。在过去的三年里，我设计并描述了一种 使用计算蛋白质设计方法和生物物理技术的人造蛋白质生物传感器。这部作品 是蛋白质-蛋白质界面中小分子结合部位从头设计的第一个例子 一个功能齐全的模块化传感器/执行器系统。在这项提议中，我的目标是建立一个新的平台 变构蛋白的特性和工程信号转导。在我的合作导师Dr。 苏珊·马尔库塞，氢交换方法的先驱，我在目标1中建议映射变构和能量 Lac阻遏物(LaC)中蛋白质残基、配体和溶剂之间的氢氚偶联 交换质谱仪(HX/MS)和等温滴定量热法。在Dr。 Kortemme，我将通过重新设计Laci来计算测试这个机制，以将其变构反应切换到 不同的配体，建立了一种控制蛋白质构象平衡的策略。在目标2中，我建议 将这种方法扩展到LacI/GalR糖反应转录因子家族中的蛋白质，以确定 并验证变构在结构和功能相关的蛋白质中保守到什么程度。在AIM 3、我将应用我们的平台来刻画变构在一个古老、高度 保守的蛋白质，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，以及加州大学旧金山分校和加州大学伯克利分校更好的研究环境，我将接受HX/MS培训， 计算方法的发展，大数据集的定量分析，库的构建，以及高 吞吐量筛选。这些技能将帮助我在生物化学工程方面的背景与我的 痴迷于蛋白质调控，建立了一个成功的独立研究计划，研究 管理变构的基本原理，以及设计新的治疗性蛋白质。