Probe the conformational dynamics of a protein targeting machine at single molecule resolution

观察分子机器的动态运动并破译其在生物功能中的作用是生命科学研究的前沿。该项目旨在阐明蛋白质靶向机器，信号识别粒子（SRP）的分子运动，因为它将约30%的新合成蛋白质传递到细胞中正确的生物膜上。该项目将产生对许多其他研究人员有用的有价值的试剂、工具和分析方法。研究结果将通过学术期刊的出版物以及向公众提供的动画电影插图进行传播。该提案的教育部分将强调培养多学科生物化学和生物物理研究方面的研究生和博士后学者。它还将使高中生和本科生接触到最先进的研究工具，并为研究生和博士后提供领导经验。更具体地说，该项目将结合Shan实验室在机械生物化学方面的专业知识和Weiss实验室在生物物理学方面的专业知识，以破译SRP在单分子分辨率上驱动共翻译蛋白靶向的动态运动。Shan实验室最近的工作表明，在蛋白质靶向周期中，SRP中存在多个大规模的构象重排。本项目将开发基于溶液的单分子荧光分析，直接观察SRP在靶向过程中的构象重排，破译驱动它们的分子力，并通过SRP的GTPase周期和通路中的空间和时间信号来了解它们的调控。这些结果将阐明这种靶向机器的时空控制的分子基础，并揭示生物学中核苷酸驱动的大分子机器作用的可推广原理。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

Observing the dynamic movements of molecular machines in action and deciphering their roles in biological function are the frontiers of life science research. This project aims to elucidate the molecular movements in a protein targeting machine, the signal recognition particle (SRP), as it delivers ~30% of newly synthesized proteins to the correct biological membrane in the cell. The project will generate valuable reagents, tools, and assays that are useful to many other researchers. The results of the research will be disseminated through publications in academic journals as well as animated movie illustrations that will be available to the general public. The education component of the proposal will emphasize the training of graduate students and postdoctoral scholars in multidisciplinary biochemical and biophysical research. It will also expose high school and undergraduate students to state-of-the-art research tools and provide leadership experience for the graduate students and postdocs.More specifically, this project will combine the expertise of the Shan lab in mechanistic biochemistry and the Weiss lab in biophysics to decipher the dynamic motions that drive co-translational protein targeting by SRP at single molecule resolution. The recent works by the Shan lab indicated the presence of multiple largescale conformational rearrangements in SRP during the protein targeting cycle. This project will develop solution-based single molecule fluorescence assays to directly observe the conformational rearrangements in SRP during targeting, decipher the molecular forces that drive them, and understand their regulation by the GTPase cycle of SRP and by spatial and temporal signals in the pathway. The results will elucidate the molecular basis for the spatiotemporal control of this targeting machine, and reveal generalizable principles that underlie the action of nucleotide-driven macromolecular machines in biology.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.