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.

项目摘要 研究策略 分子生物医学研究中的两个最大挑战是（1）缺乏详细的技术， 研究蛋白质在细胞或天然环境中的构象，和（2）缺乏蛋白质的数据 大蛋白质和跨时间尺度的构象动力学。该提案同时解决了这两个重大问题 通过开发两种密切相关的技术来迎接挑战：高通量二硫键扫描， 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 在蛋白质内的数千个残基对之间以大规模平行的形式进行波动。我不仅会 发展这些强大的技术，但也将其应用于关键的生物医学问题：构象的作用， 分子伴侣功能的可塑性;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。 候选人与环境 我是一名训练有素的实验蛋白质生物化学家和生物药剂学家， 二硫键和氧化还原化学在蛋白质结构、稳定性、错误折叠、聚集中的作用， 疾病的发病机制。通过进行拟议的研究，我将获得关键的技术专长， 新兴的蛋白质测序技术和计算建模和数据分析算法，以及 统计物理学，微生物生理学和实验进化的更深入的知识。哈佛大学 大波士顿地区的学术界为学术专业人士提供了一流的环境， 发展已征聘一批世界领先的专家担任咨询委员会成员。主 Mentor在顶尖研究密集型机构培训未来教师方面有着良好的记录。我会 充分利用职业发展课程、关于包容性教学和建设社区的讲习班 归属感，领导研究小组的研讨会，指导和实践指导，以及正式的复习 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 (14出版物在8年，开始作为一个本科生），并从这个奖项产生的工作将广泛 传播和分享。