(338b) Tandem Membrane Protein Cryo-EM and Assays in Dynamically Loadable and Intact Lipid Bilayers in Nanoporous Membranes

The objective of this research is the development of a nanoporous membrane-supported biomembrane platform to enable cryo-EM studies of membrane proteins in intact biomembranes that is not currently possible. This architecture is open to the loading of relevant membrane proteins (MPs), substrates and binding partners by lateral diffusion and can be combined with functional protease assays in the same intact biomembrane under cryo-EM study. Thus far, cryo-EM of MPs in intact biomembrane systems are prepared in batch and are not accessible via lateral diffusion, this includes MPs in nanodiscs and other recent work in liposomes. Nanoporous supported systems provide a unique solid structure to present MPs in for cryo-EM investigation in controlled orientations and in-membrane concentrations. Through this proposed technology we build open systems accessible to lateral diffusion-based loading where MP-MP and MP-soluble protein interactions can conceivably be staged in the cryo-EM observation volume to look at complex formation in intact lipid bilayers. And furthermore, phase separation is staged in the same volume allowing for studies of the direct effect of biomembrane thickness on MP structure and MP-MP complex formation in situ, and both of these types of studies are currently very difficult to stage in the cryo-EM observation region.

We have coupled the cryo-EM studies in the observation region with AFM and fluorescence imaging microscopy at both the micro- and nanoscale in these systems, using the imaging to examine to bottom-up construction of the supported lipid bilayers. Silane-PEG-NH2 tethering was used to functionalize the nanoporous silicon nitride and silica EM surfaces and we examined the coverage using AIRYSCAN superresolution with appropriate controls. We measured the lateral mobility of the lipids within these systems using confocal fluorescence recovery after photobleaching (FRAP), finding that the lipid diffusivities are within the expected values consistent with high quality supported bilayers. We probed these same bilayers with cryo-EM and showed that high quality bilayers were indeed positioned within the observation region. We followed up these measurements with detailed atomic-force microscopy (AFM) studies of coverage and supported membrane thickness, which we have recently used to image MPs such as gamma-secretase at the single molecule level in intact lipid bilayers with biomembrane phase separation(Barros et al. (2020) Langmuir 36, 23, 6569–6579).

We conclude that we formed bilayers with complete and mobile coverage that are well-suited for the introduction of membrane proteins within the cryo-EM observation zone. This method for direct imaging studies of membrane proteins in intact biomembranes of controlled composition can significantly enable a new window of structural biology of these drug targets.