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Abstract The organization of membrane proteins between and within membrane-bound compartments is critical to cellular function. Yet we lack approaches to regulate this organization in a range of membrane-based materials, such as engineered cells, exosomes, and liposomes. Uncovering and leveraging biophysical drivers of membrane protein organization to design membrane systems could greatly enhance the functionality of these materials. Towards this goal, we use de novo protein design, molecular dynamic simulations, and cell-free systems to explore how membrane-protein hydrophobic mismatch could be used to tune protein cotranslational integration and organization in synthetic lipid membranes. We find that membranes must deform to accommodate membrane-protein hydrophobic mismatch, which reduces the expression and co-translational insertion of membrane proteins into synthetic membranes. We use this principle to sort proteins both between and within membranes, thereby achieving one-pot assembly of vesicles with distinct functions and controlled split-protein assembly, respectively. Our results shed light on protein organization in biological membranes and provide a framework to design self-organizing membrane-based materials with applications such as artificial cells, biosensors, and therapeutic nanoparticles. 

Abstract Targeted vesicle fusion is a promising approach to selectively control interactions between vesicle compartments and would enable the initiation of biological reactions in complex aqueous environments. Here, we explore how two features of vesicle membranes, DNA tethers and phase‐segregated membranes, promote fusion between specific vesicle populations. Membrane phase‐segregation provides an energetic driver for membrane fusion that increases the efficiency of DNA‐mediated fusion events. The orthogonality provided by DNA tethers allows us to direct fusion and delivery of DNA cargo to specific vesicle populations. Vesicle fusion between DNA‐tethered vesicles can be used to initiate in vitro protein expression to produce model soluble and membrane proteins. Engineering orthogonal fusion events between DNA‐tethered vesicles provides a new strategy to control the spatiotemporal dynamics of cell‐free reactions, expanding opportunities to engineer artificial cellular systems.