Probing Complexity in Protein Structural Landscapes by Fluorescence and Microfluidics

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Proteins have evolved to assume a large degree of complexity encoded in a multidimensional energy landscape. Given their functional roles in cells, it is of the utmost importance to achieve a detailed understanding of these aspects. The development of enhanced experimental methods can open the door to revealing previously unavailable information critical to such an understanding. In this project, to study the physics of topological, dynamic and other structural complexity in protein systems, a combination of novel high-resolution fluorescence microscopy and microfluidic technologies will be utilized. Advances will include developments in a new experimental technique enabling rapid cooling of a small volume of protein solution for high resolution kinetic studies. These techniques in combination with single-molecule Förster resonance energy transfer (smFRET) microscopy will be applied to some key problems in protein biophysics, including investigations of complexity in protein knotting, symmetry-related effects, and non-equilibrium dynamics. The experimental research will also be linked with theoretical and computational analysis to further enhance the level of insight gained. The research is expected to lead to the development of a new set of advanced experimental tools and provide important information on protein structural complexity, with broad implications for protein function in cells and organisms.Broader impacts of the project are anticipated in multiple directions. Students and a post-doctoral researcher will be trained in emerging multidisciplinary methods and their application to molecular biophysics. Involvement in educational efforts that are related to the scientific research in the project will be continued. The results of the research will be disseminated through publication of papers in peer-reviewed journals and seminar presentations. Efforts to broaden participation, including through a TSRI outreach program, will also be continued. Finally, it is anticipated that the new experimental techniques and insights generated in the proposed work will have wide utility and appeal to the Biophysics and Biology communities. The synergy between the labs of the investigators, with their complementary areas of expertise in cutting-edge single-molecule biophysics and microfluidics research, will be particularly beneficial for broader impacts. Overall, this multidisciplinary project is aimed at providing enhanced experimental methods and will lead to new fundamental insights in the physics of protein complexity, while also integrating broader impacts in training, dissemination, and infrastructure.This project is being supported jointly by Biomolecular Dynamics, Structure and Function in MCB, and the Physics of Living Systems Program in the Physics Division.