CAREER: Mechanisms Controlling Signaling Reactions on Membranes through Molecular Assembly at Multiple Length Scales

职业：通过多长度尺度的分子组装控制膜上信号反应的机制

• 批准号: 2145852
• 负责人: YOUNGKWANG LEE
• 金额: $ 101.06万
• 依托单位: San Diego State University Foundation
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-03-01 至 2027-02-28
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=2145852&HistoricalAwards=false
• 关键词: CAREER; Mechanisms; Controlling; Signaling; Reactions

This award is funded in whole or in part under the American Rescue Plan Act of 2021 (Public Law 117-2). Cells rely on sophisticated biochemical reactions to signal the initiation of essential functions. Understanding the controls that ensure that these biochemical reactions occur in the right place at the right time is fundamental. In many cases, the signaling is carried out by proteins that interact and assemble into functional complexes at the membrane surface. However, our understanding of these processes is limited. The Lee lab uses a combination of biochemistry and biophysical techniques to unveil how molecular assembly modulates biochemical reactions. The proposed research has significant and broad implications, from advancing our fundamental knowledge of numerous biological processes to improving therapeutic strategies for disease. Integral to this research is an education and broader impacts program that provides authentic science learning experiences to students, including those from historically underrepresented minority groups. The planned activities include (1) developing smartphone optical microscopes for implementing course-based undergraduate research experiences (CUREs) that tackle real-world problems, (2) providing personalized research mentorships for undergraduate students, and (3) collaborating with area science teachers to develop science laboratory materials and curriculum for students in high-needs schools in San Diego County and beyond. American Rescue Plan funding is used to support this early career investigator at a critical stage in his career.Protein assembly, such as dimerization, clustering, and micro/macroscopic phase separation appears to enhance sensitivity and specificity in noisy cellular environments. However, much remains to be understood about how these processes alter the structure and function of proteins to produce the desired signaling outcome. This project focuses on Raf kinases, major signaling proteins in eukaryotic cells. The central hypothesis is that activation of Raf is regulated by different modes of molecular assembly at membrane surfaces. To test this hypothesis, the Lee lab will reconstitute Raf signaling on a supported lipid bilayer and manipulate molecular assembly at multiple length scales, from structurally well-defined complex formation to macroscopic liquid-liquid phase separation (LLPS) of proteins. Single-molecule fluorescence microscopy and time-resolved fluorescence spectroscopy will be used to quantify membrane binding, molecular diffusion, enzymatic activity, and conformational dynamics. The research will provide new insights into how cytosolic signaling molecules utilize multiscale molecular assembly to define specificity and sensitivity of biochemical reactions at the membrane surfaces. As protein assembly is a recurring and emerging theme in signal transduction, the experimental approach and theoretical framework developed in this project can be readily adapted to other membrane signaling reactions.This award was funding jointly by the Molecular Biophysics and Cellular Dynamics and Function Programs of MCB.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.

该奖项全部或部分根据2021年美国救援计划法案（公法117-2）资助。细胞依靠复杂的生化反应来发出启动基本功能的信号。了解确保这些生化反应在正确的时间发生在正确的地点的控制是至关重要的。在许多情况下，信号传导是由蛋白质进行的，这些蛋白质在膜表面相互作用并组装成功能复合物。然而，我们对这些过程的理解是有限的。Lee实验室使用生物化学和生物物理技术的组合来揭示分子组装如何调节生化反应。这项研究具有重大而广泛的意义，从推进我们对许多生物过程的基础知识到改善疾病的治疗策略。这项研究的组成部分是一个教育和更广泛的影响计划，为学生提供真实的科学学习经验，包括那些历史上代表性不足的少数群体。计划的活动包括（1）开发智能手机光学显微镜，用于实施基于课程的本科生研究经验（CURES），解决现实世界的问题，（2）为本科生提供个性化的研究指导，以及（3）与地区科学教师合作，为圣地亚哥县及其他地区高需求学校的学生开发科学实验室材料和课程。美国救援计划基金用于支持这位早期职业研究者在其职业生涯的关键阶段。蛋白质组装，如二聚化，聚类和微观/宏观相分离似乎可以提高嘈杂细胞环境中的灵敏度和特异性。然而，关于这些过程如何改变蛋白质的结构和功能以产生所需的信号结果，仍有许多问题有待了解。本项目的重点是Raf激酶，真核细胞中的主要信号蛋白。中心假设是Raf的活化受膜表面分子组装的不同模式调节。为了验证这一假设，Lee实验室将在支持的脂质双层上重建Raf信号，并在多个长度尺度上操纵分子组装，从结构上明确的复合物形成到蛋白质的宏观液液相分离（LLPS）。单分子荧光显微镜和时间分辨荧光光谱将用于定量膜结合，分子扩散，酶活性和构象动力学。这项研究将为胞质信号分子如何利用多尺度分子组装来定义膜表面生化反应的特异性和敏感性提供新的见解。由于蛋白质组装是信号转导中反复出现的主题，该项目中开发的实验方法和理论框架可以很容易地适用于其他膜信号传导反应。该奖项由MCB的分子生物物理学和细胞动力学和功能计划联合资助。该奖项反映了NSF的法定使命，并被认为值得通过利用基金会的智力价值进行评估来支持和更广泛的影响审查标准。