CAREER: Mechanical unfolding and refolding of multidomain proteins as a new signaling mechanism, studied using a novel single molecule approach

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This project will improve our understanding of how proteins function in vivo under force. Proteins are a major component of tissues and organs and, to perform their function, they acquire a specific 3-dimentional structure via a process known as folding. A key mechanism regulating the functioning of proteins may be the use of their unfolding/refolding transitions in response to their biomechanical environment. Little is currently known about this process, as its investigation requires novel experimental approaches that can reproduce the force experienced by individual proteins. This CAREER project aims to look beyond the established view of protein folding as a mean to acquire a characteristic 3-dimentional structure and show that proteins operating under force use domain unfolding and refolding in vivo regularly. Understanding these processes will have widespread consequences in comprehending the normal functioning of tissues. Protein folding, as a mechanism of function, is a multifaceted topic, wherein biology meets chemistry, engineering and physics. Educational and outreach activities, integrated with the proposed research goals, include: establishing a new science and technology exhibit, which will explain how protein unfolding and refolding works at molecular level; improving and expanding a biophysics internship program for K-12 students; introducing new graduate-level courses in biophysics and related disciplines; and implementing computer programing as a new "second language" for undergraduate students. The PI will engage students in outreach and education activities from several areas of science, while encouraging participation of women and underrepresented minorities.This project will investigate the relation between the structure, function and unfolding and refolding dynamics of proteins under force and how this process can swiftly change the stiffness of a tissue, perform useful mechanical work, efficiently store and release energy, and act as an on/off switch to recruit and release ligands. The PI will take advantage of a novel single molecule approach based on magnetic tweezers and covalent attachment. Using this approach, single protein molecules can be exposed to force protocols resembling those experienced in vivo, for up to 15 days, and in the presence of various buffers. This project aims to establish the rules of how protein unfolding and refolding under force determine the behavior of several important systems in nature. Furthermore, this project will provide biomedical researchers with a new perspective on the relation between protein folding and function, which will likely spur development of new therapeutic interventions.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.