High-throughput disulfide and FRET scanning to reveal protein conformational ensembles in vitro and in vivo.

高通量二硫键和 FRET 扫描可揭示体外和体内蛋白质构象整体。

• 批准号: 10191303
• 负责人: Evgeny Serebryany
• 金额: $ 10万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-10 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10191303
• 关键词: Address; Advisory Committees; Affect; Affinity Chromatography; Algorithmic Analysis; Antibiotic Resistance; Antibiotic susceptibility; Antibiotics; Aprotinin; Area; Award; Bar Codes; Binding Proteins; Biological Assay; Biomedical Research; Biophysics; Boston; Cells; Chemicals; Chemistry; Chromosomes; Communities; Computational algorithm; Computer Models; Crystallization; Cytolysis; Data; Data Analyses; Development; Disease; Disulfides; Educational process of instructing; Educational workshop; Environment; Escherichia coli; Evolution; Fluorescence Resonance Energy Transfer; Frequencies; Future; Future Teacher; Gene Library; Genotype; Goals; Head; In Vitro; Institution; Investigation; Ions; Kinetics; Knowledge; Lasers; Libraries; Light; Location; Maps; Mass Spectrum Analysis; Medical; Mentors; Mentorship; Methods; Microbial Physiology; Molecular; Molecular Chaperones; Molecular Conformation; Oxidation-Reduction; Pathogenesis; Pathway interactions; Penicillin-Binding Proteins; Peptide Sequence Determination; Peptides; Peptidyltransferase; Pharmaceutical Preparations; Phase; Phenotype; Physics; Physiologic pulse; Point Mutation; Positioning Attribute; Productivity; Protein Conformation; Protein Dynamics; Proteins; Publications; Research; Research Project Summaries; Ribonucleases; Role; Scanning; Seminal; Site; Slide; Source; Spottings; Staphylococcus aureus; Structure; Sulfhydryl Compounds; Technical Expertise; Techniques; Technology; Testing; Time; Training; Tube; Universities; Variant; Work; base; career; career development; community building; conformer; course development; design; dimer; disulfide bond; experience; experimental study; fitness; high throughput technology; improved; in vivo; member; monomer; next generation; non-Native; novel therapeutics; oxidation; pathogenic bacteria; periplasm; protein complex; protein structure; recruit; research and development; resistance mutation; responsible research conduct; single molecule; single-molecule FRET; structural biology; tool; undergraduate student

PROJECT SUMMARY Research Strategy Two of the biggest challenges in molecular biomedical research are (1) the lack of technologies for detailed investigation of protein conformations in the cell or the native environment, and (2) a dearth of data on protein conformational dynamics for large proteins and across time scales. This proposal addresses both these grand challenges head-on by developing two closely related technologies: high-throughput disulfide scanning, which will bridge the gap between in vitro and in vivo structural biology by revealing protein structures in both contexts, and single-molecule FRET scanning, which will reveal both average conformation and pairwise distance fluctuations across thousands of residue pairs within a protein in a massively parallel format. I will not only develop these powerful techniques, but also apply them to key biomedical questions: the role of conformational plasticity in the function of chaperones; the role of conformational dynamics in antibiotic susceptibility of bacterial transpeptidases; phenotypic effects of kinetically trapped and non-native protein conformations; and structural basis of genotype-phenotype relationships. Candidate and Environment I am a highly trained experimental protein biochemist and biophysicist with extensive experience investigating the roles of disulfide bonds and redox chemistry in protein structure, stability, misfolding, aggregation, and the pathogenesis of disease. By carrying out the proposed research, I will acquire crucial technical expertise in emerging protein sequencing technologies and computational modeling and data analysis algorithms, as well as a deeper knowledge of statistical physics, microbial physiology, and experimental evolution. Harvard University and the greater Boston-area academic community provides a stellar environment for academic professional development. A group of world-leading experts has been recruited as the Advisory Committee. The primary mentor has a proven track record of training future faculty members at top research-intensive institutions. I will take full advantage of career-development courses, workshops on inclusive teaching and building communities of belonging, seminars on leading a research group, guidance and practice in mentorship, and a formal refresher on responsible conduct of research. I have maintained a high level of productivity throughout my research career (14 publications in 8 years, starting as an undergraduate), and the work arising from this award will be widely disseminated and shared.

项目概要 研究策略 分子生物医学研究的两个最大挑战是（1）缺乏详细的技术 研究细胞或天然环境中的蛋白质构象，以及 (2) 缺乏蛋白质数据 大蛋白质和跨时间尺度的构象动力学。该提案解决了这两大问题 通过开发两项密切相关的技术来应对正面挑战：高通量二硫化物扫描， 将通过揭示两种情况下的蛋白质结构来弥合体外和体内结构生物学之间的差距， 和单分子 FRET 扫描，这将揭示平均构象和成对距离 以大规模并行格式研究蛋白质内数千个残基对的波动。我不仅会 开发这些强大的技术，并将它们应用于关键的生物医学问题：构象的作用 伴侣功能的可塑性；构象动力学在细菌抗生素敏感性中的作用 转肽酶；动力学捕获和非天然蛋白质构象的表型效应；和结构 基因型-表型关系的基础。 候选人和环境 我是一位训练有素的实验蛋白质生物化学家和生物物理学家，拥有丰富的研究经验 二硫键和氧化还原化学在蛋白质结构、稳定性、错误折叠、聚集和 疾病的发病机制。通过进行拟议的研究，我将获得以下领域的关键技术专业知识： 新兴的蛋白质测序技术和计算建模和数据分析算法，以及 对统计物理学、微生物生理学和实验进化有更深入的了解。哈佛大学 大波士顿地区的学术社区为学术专业人士提供了良好的环境 发展。聘请了一批世界领先的专家作为顾问委员会。初级 导师在顶级研究密集型机构培训未来教师方面拥有良好的记录。我会 充分利用职业发展课程、包容性教学和社区建设研讨会 归属感、领导研究小组研讨会、导师指导和实践以及正式复习 负责任地进行研究。在我的研究生涯中我一直保持着高水平的生产力 （从本科生开始，8年内发表14篇论文），该奖项产生的作品将被广泛传播 传播和共享。