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.

项目概要/摘要 双组分系统是细菌用来感知不同刺激的主要信号传导途径，例如 温度、渗透压变化和抗生素并启动适应性反应。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反应调节器，从而改变 基因表达。作为加州大学William DeGrado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建体来研究结构和构象 香港的动态。在这里，我建议使用结合细菌报告基因的综合实验方法， 酶测定、X 射线结构研究和基于液滴的微流体技术，以了解如何 组氨酸激酶的构象转变促进信号转导。最近的香港结构表明 二聚体界面的对称性与催化状态密切相关。该提案的目标是 开发结构信号如何诱导对称到不对称构象的分子描述 HK 催化细胞质区域的转变。我假设二聚体界面的双稳定性 “主干”赋予了必要的构象灵活性，从而允许发生局部螺旋屈曲。这 这种设计的结果是从沿着对称结构主干的连续螺旋路径过渡 状态到不连续路径，从而产生不对称性。在目标 1 中，我将研究残留物如何变化 骨干的屈曲区域影响 HK 信号传输。在目标 2 中，我将使用蛋白质设计来确定 与 HK 细胞质区域信号传导相关的结构状态。在目标 3 中，我研究了如何发出信号 传输到 HK 细胞质区域的信号通过效应域和 催化核心。完成这些目标将提供结构和结构的分子描述。 HK 的构象动力学。更好地理解结构状态和构象变化 与信号传导相关的信息可以为小分子抑制剂的基于结构的设计提供信息。通过执行 在这个提案的研究中，我将提高我在蛋白质设计和生物物理学方面的熟练程度，同时 接受 X 射线晶体学、微流体学和高通量生物学方法方面的严格培训。 这些领域的专业知识将使我能够更好地追求我的长期科学目标，即利用蛋白质设计作为 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博士和我在K99阶段获奖的合作者将帮助我变得更强 科学家，让我更好地为独立研究员的职业生涯做好准备。数据和出版物 完成本提案中的工作的结果将使我成为一名更强大的教师候选人，并有助于获得未来的资助 应用程序。一般来说，完成我的申请中提出的研究和培训将为我提供 具备实现成为首席研究员的长期职业目标所需的技能。