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

项目总结 研究策略 分子生物医学研究中最大的两个挑战是：(1)缺乏详细的 研究细胞或自然环境中的蛋白质构象，以及(2)缺乏关于蛋白质的数据 大蛋白质和跨时间尺度的构象动力学。这项建议既解决了这两个重大问题 通过开发两项密切相关的技术来正面挑战：高通量二硫化物扫描， 将通过揭示体外和体内的蛋白质结构来弥合体外和体内结构生物学之间的差距， 以及单分子FRET扫描，将同时显示平均构象和成对距离 蛋白质内数千个残基对以大规模平行的形式波动。我不仅会 开发这些强大的技术，但也将它们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；以及结构 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物理学家，具有丰富的研究经验。 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。通过开展拟议的研究，我将在以下方面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 对统计物理、微生物生理学和实验进化有更深入的了解。哈佛大学 而大波士顿地区的学术社区为学术专业人士提供了一个极好的环境 发展。已聘请一批世界领先的专家担任咨询委员会。初级阶段 Mentor拥有在顶尖研究密集型机构培训未来教职员工的良好记录。这就做 充分利用职业发展课程、包容性教学研讨会和建设社区 关于归属感、关于领导研究小组的研讨会、指导和实践，以及正式的进修 关于负责任的研究行为。在我的整个研究生涯中，我一直保持着高水平的生产力 (8年内发表14篇文章，从本科开始)，由这个奖项产生的工作将广泛 传播和分享。

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.