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

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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.