Biophysical Society Thematic Meeting | Hamburg 2022

Biophysics at the Dawn of Exascale Computers

Poster Abstracts

56-POS Board 56 SYNTHESIS OF LARGE, LIPID MEMBRANES CONTAINING MEMBRANE PROTEINS FROM GAS PHASE Matthias S Wilm 1 ; 1 University College Dublin, Conway Institute, Dublin, Ireland Objective The aim was to synthesise transportable protein containing membranes of an inherently unlimited size using a nano-electrospray ion source. Methods A nano-electrospray ion source was directed towards the liquid meniscus of a 3 mm wide reservoir holding an aqueous buffer and some SM-2 Biobeads for detergent extraction. In a series of steps bipolar lipids, glycerol and detergent solubilised membrane proteins were sprayed onto the surface and incubated to remove the detergent. Results Finally, the correct conditions were found to allow for the self-assembly of a single lipid bio-membrane containing the proteins. The layer covered the entire surface as verified by transmission electron microscopy. No denatured protein was found. Instead, the images correspond to a protein filled membrane. The protein initially used was the integral membrane protein ompG, a mono-molecular pore. Additionally I used listeriolysin O to investigate whether the procedure is gentle enough to allow non-covalent complex formation. Listeriolysin O assembles on membranes into ring shaped complexes of 30 or more units. These rings insert themselves into membranes and form large pores. Using this method the protein complex assembly process could be directly visualised. Conclusions With the help of nano-electrospray it is possible to synthesise large, transportable lipid bi-layers that contain intact membrane proteins. This is very helpful to speed up cryo-electromicroscopic structure reconstruction. The method has the potential to make the sophisticated enzymatic activity of membrane proteins accessible on a larger scale and in a technical environment, to build bio-similar detectors or enable complex molecular synthesis in large membrane filled bio- reactors. References 1. M. Wilm, bioRxiv, (https://www.biorxiv.org/content/10.1101/661215v1) 2. M. Wilm, bioRxiv, (https://www.biorxiv.org/content/10.1101/661231v1)

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