Biophysical Society Conference | Tahoe 2024

Molecular Biophysics of Membranes

Wednesday Speaker Abstracts

A HIGH-THROUGHPUT PROTEOME-WIDE PLATFORM FOR CAPTURING MEMBRANE PROTEINS IN THEIR NATIVE ENVIRONMENT FOR STRUCTURAL AND FUNCTIONAL STUDIES Caroline Brown 1,2 ; Snehasish Ghosh 1,2 ; Yansheng Liu 3,4 ; Moitrayee Bhattacharyya 3 ; Kallol Gupta 1,2 ; 1 Yale University, Cell Biology, New Haven, CT, USA 2 Yale University, Nanobiology Institute, New Haven, CT, USA 3 Yale University, Pharmacology, New Haven, CT, USA 4 Yale University, Cancer Biology Institute, New Haven, CT, USA The native membrane's local molecular environment significantly shapes membrane protein (MP) biology. However, the prevalent method, use of detergent-like molecules for MP study, removes this crucial local context, hindering our quantitative understanding of how the local bilayer governs MP structure, function, and biogenesis. Addressing this, we generated a library of membrane active polymers (MAPs) that enable nanoscale spatially-resolved extraction of MPs, directly from endogenous membranes, into native nanodiscs while maintaining the local nanoscale membrane environment. We then developed a label-free quantitative proteomics workflow to assess protein-specific extraction efficiency directly into native MAPdiscs. Leveraging this, we created a proteome-wide quantitative screening platform, reporting extraction efficiency of individual MAPs from our library in a protein-specific manner. Applying this platform to mammalian cells, using 15 different MAPs, we built a quantitative guide that reports the most optimized extraction condition of ~ 2500 unique MPs, directly from their physiological membranes. We've packaged this data into an open-access database, facilitating gene-name-specific searches yielding optimal MAP-extraction conditions for efficient extraction of target MPs or even multi-protein complexes into native nanodiscs. Using our database, we demonstrated the applicability of this platform by extracting and purifying structurally and functionally diverse MPs, directly from various organellar membranes, such as plasma membrane, endoplasmic reticulum, mitochondria, lysosome, Golgi, and even transient organelles such as autophagosome. Together, this provides a broadly applicable, rapid, and efficient avenue to capture target MPs, within local membrane ‘nano-domains’, directly from endogenous organellar membranes, maintaining physiological expression levels. We envision interfacing this platform with diverse bioanalytical approaches like MS-based OMICS, single-molecule microscopy, EM-imaging, and biochemical assays generating a transformative tool for membrane biology by offering a quantitative molecular view of the local membrane context and its regulatory impact on MPs.

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