Assembling transmembrane proteins on organic electronic materials is one promising approach to couple biological functions to electrical readouts. A biosensing device produced in such a way would enable both the monitoring and regulation of physiological processes and the development of new analytical tools to identify drug targets and new protein functionalities. While transmembrane proteins can be interfaced with bioelectronics through supported lipid bilayers (SLBs), incorporating functional and oriented transmembrane proteins into these structures remains challenging. Here, we demonstrate that cell-free expression systems allow for the one-step integration of an ion channel into SLBs assembled on an organic conducting polymer, poly(3,4-ethylenedioxythiophene) polystyrenesulfonate (PEDOT:PSS). Using the large conductance mechanosensitive channel (MscL) as a model ion channel, we demonstrate that MscL adopts the correct orientation, remains mobile in the SLB, and is active on the polyelectrolyte surface using optical and electrical readouts. This work serves as an important illustration of a rapidly assembled bioelectronic platform with a diverse array of downstream applications, including electrochemical sensing, physiological regulation, and screening of transmembrane protein modulators.

Cyclodextrin molecules are increasingly being used in biological research and as therapeutic agents to alter membrane cholesterol content, yet there is much to learn about their interactions with cell membranes. We present a biomembranebased organic electronic platform capable of detecting interactions of cell membrane constituents with methyl-β-cyclodextrin (MβCD). This approach enables label-free sensing and quantification of changes in membrane integrity resulting from such interactions. In this work, we employ cholesterol-containing supported lipid bilayers (SLBs) formed on conducting polymercoated electrodes to investigate how MβCD impacts membrane resistance. By examining the outcomes of MβCD interactions with SLBs of varying cholesterol content, we demonstrate that changes in membrane permeability or resistance can be used as a functional measure for predicting cyclodextrin-mediated cholesterol extraction from cellular membranes. Furthermore, we use the SLB platforms to electronically monitor cholesterol delivery to membranes following exposure to MβCD pre-loaded with cholesterol, observing that cholesterol enrichment is commensurate with an increase in resistance. This biomembrane-based bioelectronic sensing system offers a tool to quantify the modulation of membrane cholesterol content using membrane resistance and provides information regarding MβCD-mediated changes in membrane integrity. Given the importance of membrane integrity for barrier function in cells, such knowledge is essential for our fundamental understanding of MβCD as a membrane cholesterol modulator and therapeutic delivery vehicle.

Viral envelope fusion with the host cell membrane is dependent on a specific viral fusion peptide (FP) or loop, which becomes exposed during virus entry to drive the process of membrane fusion. In coronaviruses, the FP is a highly conserved domain that sits in the center of spike protein and in SARS-CoV, is adjacent to the S2’ proteolytic cleavage site. This peptide contains a hydrophobic LLF motif, as well as several conserved negatively charged amino acids that interact with Ca2+ ions to promote membrane fusion. In this work we perform a systematic mutagenesis study of the negatively charged amino acids within the SARS-CoV fusion peptide (FP1/FP2) and combine this with molecular dynamics simulations to define the membrane interactions that regulate virus infectivity. We show that the E801/D802 amino acid pair in the SARS-CoV FP is predicted to bind to one Ca2+ ion to promote FP-membrane interaction, with a second Ca2+ ion likely pairing residue D812 with either E821 or D825. The D812/D821 residue pair promotes membrane interaction, whereas the D821/D825 is inhibitory to membrane insertion. Taken together, our results demonstrate the dynamic nature of the coronavirus FP region that likely facilitates its interactions with and insertion into the host cell membrane.Author SummaryCoronaviruses have reemerged as a highly pathogenic virus family through the rise of SARS-CoV, MERS-CoV, and more recently, SARS-CoV-2. As more transmissible variants of SARS-CoV-2 arise, it is imperative that we understand the mechanisms of CoV viral entry to enable the development of effective therapeutics. Recent reviews have suggested the repurposing of FDA-approved calcium channel blockers to treat infection by coronaviruses; however, calcium’s method of action on viral-host cell fusion events is unknown. We have found that increased calcium availability leads to increased viral infection across the CoV family, suggesting that calcium is involved in mediating the interaction between the viral fusion peptide and the host cell membrane. As such, we hypothesize that the highly conserved fusion peptide interacts directly with calcium and this interaction is required for viral entry and infection. Through mutagenesis studies of specific negatively charged residues in the fusion peptide, we have identified residues that impact viral infectivity. We have also compared the infectivity of wild-type and mutant CoV pseudoparticles in calcium-rich or -depleted environments using chelating drugs. Our data mirrors the residue coordination observed SARS-CoV-2, as both between SARS-CoV and SARS-CoV-2 FPs bind to two calcium ions. These results demonstrate the importance of Ca2+ for CoV FP function during viral entry and opens the possibility of utilizing FDA-approved calcium-blocking drugs as a treatment for COVID-19.