Membrane proteins make up a large fraction of the human proteome and are implicated in numerous biological processes. Due to their vast importance in human biology, they are frequently the target of drug therapies and are commonly utilized in biotechnology applications. However, studying membrane protein interactions and transport in their native environment has historically been challenging. Here, we use reconstituted lipid bilayer model systems to investigate individual membrane protein interactions and transport, where the proteins are confined to a two-dimensional (2D) surface either transiently or permanently. For all of this work, we use epithelial-cadherin (E-cad) adhesion proteins either to gain direct insight into the mechanisms underlying cell-cell cohesion, or to study a model membrane-associating protein. First, we used single-molecule Total Internal Reflection Fluorescence (TIRF) microscopy to test for lateral protein clustering between E-cad, when the proteins were confined to a 2D lipid bilayer surface. After showing that E-cad formed clusters on a 2D surface, we then developed a framework combining single-molecule Förster Resonance Energy Transfer (FRET) with kinetic Monte Carlo (kMC) simulations to quantify the lateral interaction kinetics mediating these clusters. Within this framework, single-molecule FRET allowed us to directly visualize binding events between E-cad molecules, while simultaneously tracking individual E-cad dynamics on the surface. Next, we developed a biomimetic cell junction model to characterize molecular binding of E-cad within cell junctions using single-molecule FRET. Within this junction model, we tested for cooperativity between E-cad lateral (cis) and adhesive (trans) interactions using multiple E-cad mutant proteins. E-cad lateral and adhesive interactions were found to be mutually cooperative, meaning one stabilizes the other, and vice-versa. Lastly, we used Convex Lens-Induced Confinement (CLiC) to measure the facilitated diffusion of individual E-cad molecules across a wide range of confinement length-scales, where E-cad was capable of transiently adsorbing to lipid bilayers on the top and bottom surfaces. Consistent with previous theoretical predictions, the effective surface diffusion was maximized at intermediate heights. A hybrid, kinetic Monte Carlo simulation approach was used to study this process in detail, and showed that facilitated diffusion can indeed result in elevated diffusion under confinement, but only under conditions where the molecule has a low affinity for the surface. These findings provided new insights into the physics underlying the search process of peripheral membrane proteins and information useful for the design of biotechnology systems.</p
