(771d) Tether Supported Biomembranes with Phase Composition and Orientation Control for Biomaterial Ligand Displays and Membrane Protein Assays
Integral membrane proteins are highly sensitive to the composition and phases within biomembrane microenvironments of a cell and have a wide variety of functionalities, ranging from cleavage to transport to cell-to-cell signaling. The proteins often contain large extramembrane domains leading to difficulties in reconstitution in isolated systems. These proteins require a confluent bilayer, proper protein and substrate orientation, and appropriate chemical environment, along with adequate spacing to relieve outside stresses on the membrane. Despite extensive studies across a variety of integral membrane proteins, a high throughput systematic assay has yet to be developed for such proteins. In these studies, a supported lipid bilayer containing tethering and cushioning moieties is developed to isolate the biologically relevant transmembrane orientations of the membrane bound enzymatic system. This work shows two biomembrane platforms used to build new multifunctional systems, planar biomembranes and also spherical proteolipobeads that facilitate high throughput analysis. We have characterized the structures, phase localization and compositions of enzymes in proteolipobead and planar systems by employing correlative optical and surface microscopy from the micro- to the nanoscale. The primary imaging modalities are superresolution microscopy and atomic force microscopy performed in tandem. This work is directed at the at the intramembrane protease gamma-secretase, an enigmatic enzyme with major public health relevance. It is proposed that a platform containing biologically active proteins in appropriate configurations and lipid compositions with proper support structures can be developed for the functional analysis of gamma-secretase and other integral membrane proteins.