(622b) Leveraging Lipid-Protein Interactions to Engineer Spatial Organization in Cell-Free Systems

Cell-free systems are powerful tools for studying and engineering biology. However, compared to a cell, the number of biological operations in cell-free systems is limited due to the lack of compartmentalization. In biological cells, membrane-bound compartments enable cells to carry out diverse reactions in parallel and in concert driven largely through the activities of integrated membrane proteins. In order to harness the functions of cellular membranes in cell-free systems, we need to better understand how to integrate and organize membrane proteins into membranes. Membranes and membrane-proteins have been shown to possess complementary physiochemical properties, allowing for proper protein sorting and function within cells. Specifically, protein transmembrane domain length and geometry have been shown to drive protein localization between and within different cellular membranes. Inspired by this, we characterized how hydrophobic mismatch between synthetic proteins and membranes could be leveraged to spatially organize proteins between and within synthetic membranes. We first characterized how hydrophobic mismatch affects the insertion and folding of cell-free expressed membrane proteins into synthetic membranes. Next, we characterized how protein hydrophobic mismatch may be leveraged as a handle to control inter- and intramembrane organization through molecular dynamic simulations, flow cytometry, and microscopy. Finally, we demonstrate that hydrophobic mismatch controls where membrane proteins localize, enabling the controlled release of encapsulated cargo and the segregation of membrane bound enzymes. This study underscores the importance of lipid-protein interactions in cell-free systems and demonstrates how biophysical features of these parts may be used to engineer new behaviors.