Dataset Information

Reconstitution and Functional Analysis of a Full-Length Hepatitis C Virus NS5B Polymerase on a Supported Lipid Bilayer.

ABSTRACT: Therapeutic targeting of membrane-associated viral proteins is complicated by the challenge of investigating their enzymatic activities in the native membrane-bound state. To permit functional characterization of these proteins, we hypothesized that the supported lipid bilayer (SLB) can support in situ reconstitution of membrane-associated viral protein complexes. As proof-of-principle, we selected the hepatitis C virus (HCV) NS5B polymerase which is essential for HCV genome replication, and determined that the SLB platform enables functional reconstitution of membrane protein activity. Quartz crystal microbalance with dissipation (QCM-D) monitoring enabled label-free detection of full-length NS5B membrane association, its interaction with replicase subunits NS3, NS5A, and template RNA, and most importantly its RNA synthesis activity. This latter activity could be inhibited by the addition of candidate small molecule drugs. Collectively, our results demonstrate that the SLB platform can support functional studies of membrane-associated viral proteins engaged in critical biological activities.

SUBMITTER: Cho NJ

PROVIDER: S-EPMC4965852 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Reconstitution and Functional Analysis of a Full-Length Hepatitis C Virus NS5B Polymerase on a Supported Lipid Bilayer.

Cho Nam-Joon NJ Pham Edward A EA Hagey Rachel J RJ Lévêque Vincent J VJ Ma Han H Klumpp Klaus K Glenn Jeffrey S JS

ACS central science 20160613 7

Therapeutic targeting of membrane-associated viral proteins is complicated by the challenge of investigating their enzymatic activities in the native membrane-bound state. To permit functional characterization of these proteins, we hypothesized that the supported lipid bilayer (SLB) can support in situ reconstitution of membrane-associated viral protein complexes. As proof-of-principle, we selected the hepatitis C virus (HCV) NS5B polymerase which is essential for HCV genome replication, and det ...[more]

PMID: 27504492

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Project description:Biomolecular devices based on photo-responsive proteins have been widely proposed for medical, electrical, and energy storage and production applications. Also, bacteriorhodopsin (bR) has been extensively applied in such prospective devices as a robust photo addressable proton pump. As it is a membrane protein, in principle, it should function most efficiently when reconstituted into a fully fluid lipid bilayer, but in many model membranes, lateral fluidity of the membrane and protein is sacrificed for electrochemical addressability because of the need for an electroactive surface. Here, we reported a biomolecular photoactive device based on light-activated proton pump, bR, reconstituted into highly fluidic microcavity-supported lipid bilayers (MSLBs) on functionalized gold and polydimethylsiloxane cavity array substrates. The integrity of reconstituted bR at the MSLBs along with the lipid bilayer formation was evaluated by fluorescence lifetime correlation spectroscopy, yielding a protein lateral diffusion coefficient that was dependent on the bR concentration and consistent with the Saffman-Delbrück model. The photoelectrical properties of bR-MSLBs were evaluated from the photocurrent signal generated by bR under continuous and transient light illumination. The optimal conditions for a self-sustaining photoelectrical switch were determined in terms of protein concentration, pH, and light switch frequency of activation. Overall, a significant increase in the transient current was observed for lipid bilayers containing approximately 0.3 mol % bR with a measured photo-current of 250 nA/cm2. These results demonstrate that the platforms provide an appropriate lipid environment to support the proton pump, enabling its efficient operation. The bR-reconstituted MSLB model serves both as a platform to study the protein in a highly addressable biomimetic environment and as a demonstration of reconstitution of seven-helix receptors into MSLBs, opening the prospect of reconstitution of related membrane proteins including G-protein-coupled receptors on these versatile biomimetic substrates.

Dataset Information

Reconstitution and Functional Analysis of a Full-Length Hepatitis C Virus NS5B Polymerase on a Supported Lipid Bilayer.

Publications

Reconstitution and Functional Analysis of a Full-Length Hepatitis C Virus NS5B Polymerase on a Supported Lipid Bilayer.

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