Biophysical studies of macromolecules and molecular assemblies

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Project summary/abstract This MIRA renewal grant proposal briefly summarizes the accomplishments over the past 4 years of support and outlines plans for continued support. The theme that unifies this research is the development and application of new physical methods that can impact the quantitative analysis of complex biological systems. The freedom to develop and broaden our research provided by the MIRA support has led to a significant evolution of the emphasis of part of our work on infectious diseases. Specifically, we will focus on the biomedically critical need to understand the origin(s) of antibiotic resistance using the TEM -lactamases as an initial target. Likewise, our efforts to develop novel ways to organize and manipulate biological membranes now focus on the mechanism of viral membrane fusion. While these two areas had completely separate origins in the parent R01’s that were merged in the MIRA, they have both provided rich areas for new and impactful research. My lab develops spectroscopic methods for probing protein-exerted electric fields which we use to obtain quantitative information on how electric fields contribute to catalysis at the active sites of enzymes. We led the development of vibrational Stark effect spectroscopy as a general approach to map these fields. Using this approach, we can, for the first time, quantify the electrostatic contribution to the catalytic proficiency of enzymes. Moving beyond ideal model enzymes, we will use this approach to provide a deeper understanding of the mechanism(s) by which TEM--lactamases evolve to cope with man-made antibiotics. By studying the connection between evolution and electric fields, we hope to develop general design principles for these enzymes and discover the physical origins of antibiotic resistance. We discovered that “split” GFPs can be photo-dissociated, and we study the underlying mechanism of this unusual process for optogenetic applications. This deeper view of strand photo-dissociation along with our work elucidating factors that control bond-specific photo-isomerization pathways are connected to our work on protein electrostatics and will provide a framework for understanding GFP’s electro-optic properties. Our lab pioneered the development of model membrane architectures, along with imaging and analytical methods that probe fundamental aspects of biological membrane organization and dynamics. Our current focus is the application of these architectures and novel single particle assays to characterize the elementary steps by which enveloped viruses, such as influenza A, fuse to target membranes. In parallel, we characterize the organization of lipids with high lateral resolution using imaging mass spectrometry. Recently we showed that atom recombination can be used to identify which lipids and proteins are in very close proximity (< 3nm) in biological membranes. This new approach addresses major challenges in membrane biophysics and structural biology where local organization is key to emergent function.