Cell-free synthesis of a transmembrane mechanosensitive channel protein into a hybrid-supported lpid bilayer

Authors: MANZER, ZACHARY - Cornell University; GHOSH, SURAJIT - Cornell University; JACOBS, MIRANDA - Northwestern University; KRISHNAN, SRINIVASAN - Boyce Thompson Institute; ZIPFEL, WARREN - Cornell University; Pineros, Miguel; KAMAT, NEHA - Northwestern University; DANIEL, SUSAN - Cornell University

Submitted to: ACS Applied Materials and Interfaces
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/19/2021
Publication Date: 3/18/2021
Citation: Manzer, Z., Ghosh, S., Jacobs, M.L., Krishnan, S., Zipfel, W.R., Pineros, M., Kamat, N.P., Daniel, S. 2021. Cell-free synthesis of a transmembrane mechanosensitive channel protein into a hybrid-supported lpid bilayer. ACS Applied Materials and Interfaces. 4(4):3101-3112. https://doi.org/10.1021/acsabm.0c01482.
DOI: https://doi.org/10.1021/acsabm.0c01482

Interpretive Summary: Proteins embedded in the cellular membranes mediate the transport of ions and nutrients that enter and leave any cell. These transport proteins are a key component in biological membranes, as they play key roles in regulating many essential biological and environmental responses. Understanding processes that take place at the cellular level requires the development of new techniques that recreate and mimic the complex microenvironment occurring at the molecular scale. In this study, we report on the development of a methodology that allows the incorporation of transmembrane proteins into a pre-assembled artificial membrane. This methodology uses a cell-free expression system containing the machinery to target and insert the translated protein into the artificial membrane. The resulting biomimetic membrane is compatible with several microscopy, biophysical, and analytical tools commonly used by scientists to understand biological phenomena occurring at this interface. We demonstrate the utility and generality of this technology in recreating and preserving the complex membrane environment for applications requiring cell membrane-like interfaces, required to understand the abiotic-biotic relations governing transport proteins structure and functionality.

Technical Abstract: Supported lipid bilayers (SLBs) hold tremendous promise as cellular-mimetic structures that can be readily interfaced with analytical and screening tools. The incorporation of transmembrane proteins, a key component in biological membranes, is a significant challenge that has limited the capacity of SLBs to be used for a variety of biotechnological applications. Here, we report an approach using a cell-free expression system for the cotranslational insertion of membrane proteins into hybrid-supported lipid bilayers (HSLBs) containing phospholipids and diblock copolymers. We use cell-free expression techniques and a model transmembrane protein, the large conductance mechanosensitive channel (MscL), to demonstrate two routes to integrate a channel protein into a HSLB. We show that HSLBs can be assembled with integrated membrane proteins by either cotranslational integration of protein into hybrid vesicles, followed by fusion of these proteoliposomes to form a HSLB, or preformation of a HSLB followed by the cell-free synthesis of the protein directly into the HSLB. Both approaches lead to the assembly of HSLBs with oriented proteins. Notably, using single-particle tracking, we find that the presence of diblock copolymers facilitates membrane protein mobility in the HSLBs, a critical feature that has been difficult to achieve in pure lipid SLBs. The approach presented here to integrate membrane proteins directly into preformed HSLBs using cell-free cotranslational insertion is an important step toward enabling many biotechnology applications, including biosensing, drug screening, and material platforms requiring cell membrane-like interfaces that bring together the abiotic and biotic worlds and rely on transmembrane proteins as transduction elements.