Biophysical Society Conference | Tahoe 2022

Molecular Biophysics of Membranes

Thursday Speaker Abstracts

DROPLET-ON-HYDROGEL BILAYER BASED ASSAY FOR FUNCTIONAL STUDY OF MEMBRANE PROTEINS Zuzana Coculova 1 ; Richard Berry 1 ; 1 University of Oxford, Department of Physics, Oxford, United Kingdom Functional study of membrane proteins, especially when transmembrane potential is required, is notoriously difficult. Various creative approaches - using either liposomes, lipid nanodiscs, or a wide range of different planar biomembrane systems - did contribute to building our understanding of these molecular machines, but the picture is not complete yet. Here we introduce a system, which adapts the droplet-on-hydrogel technique [1], for high resolution optical studies of membrane proteins with independent control of the electrical and chemical transmembrane potentials. The experiment is performed by embedding a membrane protein of interest into a lipid bilayer formed between a 200 nl water droplet and a supporting thin layer of agarose. This guarantees stability, hydration and accessibility of the membrane. The protein is delivered in positively charged proteoliposomes, which fuse with the negatively charged lipid bilayer [2]. Sample delivery into the water droplet, and continuous control over the proteins’ environment, is achieved through two glass micropipettes of our custom built electronically controlled perfusion system.We chose to demonstrate the capabilities of our assay by studying the Esterichia coli F 1 F o . This complex of two molecular motors - membrane embedded F o and water soluble F 1 - catalyses the synthesis of ATP molecules, the basic energy units of life, using the transmembrane proton gradient created during respiration or photosynthesis. Our assay will allow us to mimic native energized membranes, study activation potential at different conditions, as well as observe single-molecule rotation steps of F 1 F o at the time scale of a few microseconds. We would also like to use this opportunity to get in contact with scientists who may find our assay helpful for studying other biological systems in energized biological membranes. [1] Leptihn, S. et al. Nature Protocol. 8 (6): 1048-10572 (2013) [2] Ishmukhametov, R. et al. Nature Communications. 7: 13025 (2016)

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