Cell-free expression with a quartz crystal microbalance enables rapid, dynamic, and label-free characterization of membrane-interacting proteins

Abstract

AbstractIntegral and interacting membrane proteins (IIMPs) constitute a vast family of biomolecules that perform essential functions in all forms of life. However, characterizing their interactions with lipid bilayers remains limited due to challenges in purifying and reconstituting IIMPsin vitroor labeling IIMPs without disrupting their functionin vivo. Here, we report TXTL-QCMD to dynamically characterize interactions between diverse IIMPs and membranes without protein purification or labeling. As part of TXTL-QCMD, IIMPs are synthesized using cell-free transcription-translation (TXTL), and their interactions with supported lipid bilayers are measured using a quartz crystal microbalance with dissipation (QCMD). TXTL-QCMD reconstitutes known IIMP-membrane dependencies, including specific association with prokaryotic or eukaryotic membranes or oscillating interactions by theE. coliMin system. Applying TXTL-QCMD to the recently discovered Zorya anti-phage system unamenable to labeling, we discovered that ZorA and ZorB integrate together within the lipids found at the poles of bacteria while ZorE diffuses freely on the non-pole membrane. These efforts establish the potential of TXTL-QCMD to broadly characterize the large diversity of IIMPs.