(595c) Membrane Protein Cryo-EM and Assays in Dynamically Loadable Intact Lipid Bilayers on Nanoporous Membranes
AIChE Annual Meeting
2022
2022 Annual Meeting
Food, Pharmaceutical & Bioengineering Division
Micro- and Nano-Scale Technologies in Life Sciences II
Thursday, November 17, 2022 - 8:36am to 8:54am
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 are 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 liposomes, detergent micelles, and other recent work in nanodiscs. 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 functional assays and 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. These measurements included atomic-force microscopy (AFM) 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, 6569â6579). This method for direct imaging studies of membrane proteins in intact biomembranes of controlled composition can significantly enable a new window of structural biology and assays of these drug targets.