Mapping and rewiring protein allostery

蛋白质变构的映射和重新布线

• 批准号: 10670446
• 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/10670446
• 关键词: 6-Phosphofructokinase; 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 Gene; 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; Mutagens; 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; biophysical properties; biophysical techniques; design; disease diagnosis; experimental study; 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.

项目摘要。 生物学调节取决于蛋白质变构，其中蛋白质中一个部位的扰动会导致A 在另一个站点的功能更改。因为变构的特征挑战了我们技术的限制 能力 - 需要同时观察结构，动力学，功能和能量学的变化 蛋白质合奏 - 几乎没有原子细节可以理解变构机制。这些知识差距限制 我们对疾病中如何保存，进化或影响的天生的基本理解以及我们 设计新的变构蛋白的能力。桥接间隙需要绘制，验证和调整特定的方法 驱动蛋白质以将其等效转移到占据不同功能状态的相互作用。 我是UCSF的NIH IRACDA和UCSF校长的博士后研究员。我的精神是坦贾博士 Kortemme，计算生物物理学专家。在过去的三年中，我设计了一个 使用计算蛋白设计方法和生物物理技术的人工蛋白生物传感器。这项工作 是蛋白质 - 蛋白质界面中小分子结合位点的从头设计的第一个例子 功能性的模块化传感器/执行器系统。在此提案中，我旨在建立一个新的平台 表征和工程信号转导的变构蛋白。在我的同事博士的指导下 Susan Marqusee，氢交换方法的先驱，我在AIM 1提议绘制变构和能量 蛋白质保留，配体之间的耦合并使用氢 - 居民在LAC复制品（LACI）中求解 用质谱法（HX/MS）和等温滴定量热法交换。凭借博士的精神训练 Kortemme，我将通过重新设计LACI将其变构响应重新设计为计算测试此机制 不同的配体，建立了控制蛋白质概念平衡的策略。在AIM 2中，我建议 将这种方法扩展到糖反应转录因子的LACI/GALR家族中的蛋白质，以确定 并验证在结构和功能相关的蛋白质之间配置了何种程度的变构。目标 3，我将应用我们的平台来表征在古老的，高度 保守的蛋白质，并了解与癌症相关的突变如何影响其机制。在我的支持下 UCSF和UC Berkeley的导师，合作者以及更大的研究环境，我将在HX/MS培训， 计算方法开发，大型数据集的定量分析，图书馆构建和高级 吞吐量筛选。这些技能将有助于我与我的生化工程背景联系在一起 对蛋白质调节的迷恋，以建立成功的独立研究计划，调查 基本原理，这些原则和工程新的治疗蛋白。

项目成果

Reply to Liu et al.: Specific mutations matter in specificity and catalysis in ACE2.

• DOI: 10.1073/pnas.2024450118
• 发表时间: 2021-04-13
• 期刊: Proceedings of the National Academy of Sciences of the United States of America
• 影响因子: 11.1
• 作者: Glasgow J;Glasgow A;Kortemme T;Wells JA
• 通讯作者: Wells JA