Hydrophobic mismatch drives self-organization of designer proteins into synthetic membranes

The extent to which membrane biophysical properties, such as hydrophobic thickness, can drive membrane protein organization remains unknown. Inspired by this question, we used de novo protein design, molecular dynamic simulations, and cell-free systems to elucidate how membrane-protein hydrophobic mismatch affects protein integration and organization in synthetic lipid membranes. We found 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 used 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 self-organizing membrane-based materials with new functions.