Characterization of bacterial sensors using protein design

使用蛋白质设计表征细菌传感器

• 批准号: 9165148
• 负责人: Nathan Schmidt
• 金额: $ 9万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-22 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9165148
• 关键词: Address; Affect; Amino Acids; Anti-Bacterial Agents; Antibiotic Resistance; Antibiotics; Applications Grants; Area; Award; Bacteria; Biological Assay; Biology; Biophysics; California; Catalytic Domain; Couples; Coupling; Crystallization; Crystallography; DNA; Data; Drug Design; Drug Targeting; Drug resistance; Effectiveness; Environment; Enzymes; Equilibrium; Face; Faculty; Future; Gene Expression; Gene Proteins; Goals; Laboratories; Libraries; Maps; Measures; Membrane; Mentors; Methods; Microfluidics; Modification; Molecular; Molecular Conformation; Pharmaceutical Preparations; Phase; Phenotype; Phosphoric Monoester Hydrolases; Phosphotransferases; Population; Predisposition; Principal Investigator; Prokaryotic Cells; Proline; Property; Protein Engineering; Proteins; Publications; Reporter; Research; Research Personnel; Research Training; Roentgen Rays; San Francisco; Scientist; Shapes; Signal Pathway; Signal Transduction; Stimulus; Structure; System; Technology; Temperature; Tertiary Protein Structure; Testing; Training; Transmembrane Domain; Universities; Vertebral column; Virulence; Work; X-Ray Crystallography; abstracting; antimicrobial; bacterial resistance; base; career; combinatorial; conformational conversion; design; dimer; experience; flexibility; global health; next generation sequencing; protein structure function; protein-histidine kinase; response; sensor; skills; small molecule; small molecule inhibitor

Project Summary/Abstract Two-component systems are major signaling pathways bacteria use to sense diverse stimuli such as temperature, osmotic changes, and antibiotics and to initiate adaptive responses. In these systems the histidine kinase (HK) detects the stimulus and relays the signal to its cognate response regulator, which alters gene expression. As a postdoctoral scholar in Dr. William DeGrado's laboratory at the University of California, San Francisco (UCSF), I have been designing protein constructs to study the structural and conformational dynamics of HKs. Here, I propose to use an integrative experimental approach combining bacterial reporters, enzymatic assays, x-ray structural studies, and droplet-based microfluidics technologies to understand how conformational transitions in histidine kinases facilitate signal transduction. Recent HK structures suggest that symmetry across the dimer interface is intricately related to catalytic state. The goal of this proposal is to develop a molecular description of how structural signals induce symmetric to asymmetric conformational transitions in the catalytic, cytoplasmic regions of HKs. I hypothesize that bistability of the dimer interface `backbone' confers an essential conformational flexibility, which allows localized helical buckling to occur. The consequence of this design is a transition from a continuous helical path along the backbone in the symmetric state to a discontinuous path, which produces asymmetry. In Aim 1 I will examine how residue changes in the buckling region of the backbone affects HK signaling. In Aim 2 I will use protein design to determine the structural states associated with signaling in the cytoplasmic region of HKs. In Aim 3 I examine how signals transmitted into the cytoplasmic region of HKs become modulated by coupling between effector domains and the catalytic core. Completion of these aims will provide a molecular description of the structural and conformational dynamics of HKs. A better understanding of the structural states and conformational changes associated with signaling can inform structure-based design of small molecule inhibitors. By performing the research in this proposal, I will increase my proficiency in protein design and biophysics while simultaneously receiving strong training in X-ray crystallography, microfluidics, and high-throughput approaches to biology. Expertise in these areas will better allow me to pursue my long-term scientific goals of using protein design as a method to elucidate protein structure and function. The experience I will receive by working with my mentor Dr. DeGrado and my collaborators during the K99 phase of the award will help me to become a stronger scientist and better prepare me for a career as an independent researcher. The data and publications that result from doing the work in this proposal will make me a stronger faculty candidate and help with future grant applications. In general, completion of the research and training proposed in my application will provide me with the skill set necessary for achieving my long-term career goal of becoming a principal investigator.

项目摘要/摘要 两个组件系统是细菌使用多种刺激的主要信号通路，例如 温度，渗透变化和抗生素，并引发自适应反应。在这些系统中 组氨酸激酶（HK）检测刺激并将信号传递给其同源响应调节剂，从而改变 基因表达。作为加州大学威廉·德格拉多博士实验室的博士后学者， 旧金山（UCSF），我一直在设计蛋白质构造来研究结构和构象 HK的动力学。在这里，我建议使用结合细菌记者的综合实验方法， 酶试验，X射线结构研究和基于液滴的微流体技术，以了解如何了解 组氨酸激酶的构象转变有助于信号转导。最近的香港结构表明 二聚体界面上的对称性与催化态相关。该提议的目的是 开发一个分子描述，说明结构信号如何诱导非对称构象对称 HKS的催化，细胞质区域的过渡。我假设二聚体界面的双重性 “骨干”赋予了必不可少的构象柔韧性，可以使局部螺旋屈曲发生。这 该设计的后果是从对称中的骨架沿骨干的连续螺旋路径的过渡 状态到不连续的路径，该路径会产生不对称性。在AIM 1中，我将研究残留物如何变化 主链的屈曲区域会影响HK信号传导。在AIM 2中，我将使用蛋白质设计来确定 与HKS细胞质区域中信号传导相关的结构状态。在AIM 3中，我检查了信号如何 传播到HKS的细胞质区域通过效应域和 催化核心。这些目标的完成将提供结构和 HKS的构象动力学。更好地理解结构状态和构象变化 与信号传导相关的小分子抑制剂可以为基于结构的设计提供信息。通过执行 在此提案中的研究，我将同时提高我对蛋白质设计和生物物理学的熟练程度 接受X射线晶体学，微流体和生物学高通量方法的强大训练。 在这些领域的专业知识将更好地使我实现使用蛋白质设计作为的长期科学目标 一种阐明蛋白质结构和功能的方法。与我的导师一起工作，我将获得的经验 Degrado博士和我的合作者在奖项的K99阶段将帮助我变得更强大 科学家，更好地为我做好了独立研究人员的职业。数据和出版物 在此提案中完成工作的结果将使我成为更强大的教师候选人，并帮助未来的赠款 申请。通常，在我的申请中提出的研究和培训的完成将为我提供 凭借实现我成为首席研究员的长期职业目标所必需的技能。