Understanding Periperal Membrane Protein Interactions | BPS Thematic Meeting

Understanding Peripheral Membrane Protein Interactions: Structure, Dynamics, Function and Therapy

Wednesday Speaker Abstracts

DECIPHERING THE ORGANELLE-TARGETING SPECIFICITY OF INDUCIBLE AMPHIPATHIC HELICES THROUGH A RECOMBINANT PROTEIN PLATFORM Antonis Margaritakis 1 ; Peter J. Chung 1,2 ; 1 University of Southern California, Physics and Astronomy, Los Angeles, CA, USA 2 University of Southern California, Department of Chemistry, Alfred E. Mann Department of Biomedical Engineering, Los Angeles, CA, USA Many peripheral membrane-binding proteins critical to membrane trafficking and function contain an unstructured domain that can bind to specific membranes, with a transition into an amphipathic helix induced upon membrane association. These inducible amphipathic helices often play a critical role in organelle recognition and subsequent function by these cytosolic proteins, but the tools and techniques used to characterize affinity towards specific membranes are low-throughput and highly dependent on the solubility of the inducible amphipathic helix. Here, we introduce a modular recombinant protein platform for rapidly measuring the binding affinity of inducible amphipathic helices towards a variety of membrane compositions and curvatures using high-throughput fluorescence anisotropy measurements. Inducible amphipathic helices are solubilized with a fluorescently tagged small ubiquitin-like modifier (SUMO) protein and binding to membranes quantified by leveraging the unexpected decrease in fluorescence anisotropy upon binding, a phenomenon previously observed but not well understood. By using fluorescence anisotropy decay measurements and solution NMR experiments, we deduce that this phenomenon likely occurs due to the local increase in fluorophore motion upon binding to the membrane. We apply this approach to representative inducible amphipathic helices to determine the sequence features by which they specifically target organelles. By systematically varying membrane parameters, we identify key physiochemical features that drive selective binding of inducible amphipathic helices with the goal of predicting how sequence dictates organelle targeting within cells.

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