Project Summary/Abstract The goal of Dr. Shannon Yan's career development application is to understand the energetic and mechanistic principles that govern structural and functional switching by proteins critical for cellular regulation. Dr. Yan will begin with studying transitions of human mitotic-arrest deficient 2 protein MAD2 in the context of co-translational protein folding, and ultimately focus on its dynamic fold-switching flux established in vivo by folding effectors and chaperones. As structure dictates the function of a biomolecule, MAD2 is capable of switching folds between an inactive open state and mitotic-arrest closed state to modulate the formation of spindle assembly during mitosis—otherwise leading to detrimental chromosome instability or cancerous development. This proposal will explore the molecular details regarding MAD2 structure-function transitions, aiming to elucidate the underlying principles for the operation of protein switches: 1) Is there a default native fold, or an initial equilibrium condition, when MAD2 is first synthesized? Dr. Yan's invention of the first single-human-ribosome translation assay on optical tweezers will directly resolve in real-time the co-translational folding trajectory for a single MAD2 protein as its nascent polypeptide gradually emerges on the human ribosome surface. 2) Is the co-translational folding pathway, and thus the prevalence of one conformer over the other, influenced by folding effectors dependent on the physiological state of cells? Dr. Yan will examine how translation rates, the presence of folding effectors/chaperons, and other cellular factors that may reshape the folding energy landscape for MAD2 protein switch. Knowledge gained from her single-molecule work in the K99 phase will fuel the subsequent cell imaging studies proposed for the R00 phase: 3) How does MAD2 oscillate between different conformer distributions—both spatially and temporally—in accordance with phases of the cell cycle? The flux of open and closed MAD2 protein switches at various stages during mitosis will be characterized and correlated to other concurrent cellular events. Results obtained during this period will enable Dr. Yan to establish herself in the field of cellular biology and cellular mechanics, in which she aims to lead a research group as a tenure-track principal investigator at an academic research institute. Dr. Yan will apply a multidisciplinary approach combining biophysics, molecular and cellular biology, and single-molecule methods, together with genetic manipulations to formulate a unique research line aimed at resolving cellular dynamics and probing the associated cellular mechanics during cell division. This five-year career development program is tailored to prepare Dr. Yan for an independent scientific career. It will build on Dr. Yan's extensive background in ribosome translation and single-molecule techniques, while expanding her skill sets in molecular biology and biochemistry to investigate folding of metamorphic protein switches. Her training program will be directed by Dr. Carlos Bustamante at UC Berkeley—an internationally recognized leader in protein folding and with extensive records of mentorship. Dr. Tanaka and Dr. Legname will also join in to support Dr. Yan's research, professional growth, and her transition into independence. The long-term objective of Dr. Yan's independent research is to expand our understanding on how cells modulate biological signals to grow and multiply, and to identify the origins of regulatory deviations that lead to diseases in humans.

Project Narrative As structure dictates the function of a biomolecule, proteins that naturally transition among more than one native fold may operate as regulatory switches by adopting distinct functional folds to modulate dynamic cellular processes, as is the case for the mitotic checkpoint protein MAD2 in humans, whose defective fold-switching is often found in cancerous diseases. To grasp and further modulate protein switches inside cells requires fully understanding the co-translational folding energy landscapes that govern the pathways along which these polypeptides attempt to fold as they are being synthesized. Using single-molecule optical tweezers and an in vitro human translation system, I aim to resolve the molecular trajectories of folding for MAD2 and mutant variants as they emerge on the human ribosome in the presence of folding effectors—and complementing with the dynamic fold-switching flux of MAD2 visualized via cell imaging—to provide mechanistic insights on MAD2 protein switch repair for potential treatments in cancers and other medical applications.