Dataset Information

Genetically Encoded Spy Peptide Fusion System to Detect Plasma Membrane-Localized Proteins In Vivo.

ABSTRACT: Membrane proteins are the main gatekeepers of cellular state, especially in neurons, serving either to maintain homeostasis or instruct response to synaptic input or other external signals. Visualization of membrane protein localization and trafficking in live cells facilitates understanding the molecular basis of cellular dynamics. We describe here a method for specifically labeling the plasma membrane-localized fraction of heterologous membrane protein expression using channelrhodopsins as a case study. We show that the genetically encoded, covalent binding SpyTag and SpyCatcher pair from the Streptococcus pyogenes fibronectin-binding protein FbaB can selectively label membrane-localized proteins in living cells in culture and in vivo in Caenorhabditis elegans. The SpyTag/SpyCatcher covalent labeling method is highly specific, modular, and stable in living cells. We have used the binding pair to develop a channelrhodopsin membrane localization assay that is amenable to high-throughput screening for opsin discovery and engineering.

SUBMITTER: Bedbrook CN

PROVIDER: S-EPMC4546540 | biostudies-literature | 2015 Aug

REPOSITORIES: biostudies-literature

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Genetically Encoded Spy Peptide Fusion System to Detect Plasma Membrane-Localized Proteins In Vivo.

Bedbrook Claire N CN Kato Mihoko M Ravindra Kumar Sripriya S Lakshmanan Anupama A Nath Ravi D RD Sun Fei F Sternberg Paul W PW Arnold Frances H FH Gradinaru Viviana V

Chemistry & biology 20150723 8

Membrane proteins are the main gatekeepers of cellular state, especially in neurons, serving either to maintain homeostasis or instruct response to synaptic input or other external signals. Visualization of membrane protein localization and trafficking in live cells facilitates understanding the molecular basis of cellular dynamics. We describe here a method for specifically labeling the plasma membrane-localized fraction of heterologous membrane protein expression using channelrhodopsins as a c ...[more]

PMID: 26211362

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Project description:During infection the SARS-CoV-2 virus fuses its viral envelope with cellular membranes of its human host. The viral spike (S) protein mediates both the initial contact with the host cell and the subsequent membrane fusion. Proteolytic cleavage of S at the S2' site exposes its fusion peptide (FP) as the new N-terminus. By binding to the host membrane, the FP anchors the virus to the host cell. The reorganization of S2 between virus and host then pulls the two membranes together. Here we use molecular dynamics (MD) simulations to study the two core functions of the SARS-CoV-2 FP: to attach quickly to cellular membranes and to form an anchor strong enough to withstand the mechanical force during membrane fusion. In eight 10 μs long MD simulations of FP in proximity to endosomal and plasma membranes, we find that FP binds spontaneously to the membranes and that binding proceeds predominantly by insertion of two short amphipathic helices into the membrane interface. Connected via a flexible linker, the two helices can bind the membrane independently, yet binding of one promotes the binding of the other by tethering it close to the target membrane. By simulating mechanical pulling forces acting on the C-terminus of the FP, we then show that the bound FP can bear forces up to 250 pN before detaching from the membrane. This detachment force is more than 10-fold higher than an estimate of the force required to pull host and viral membranes together for fusion. We identify a fully conserved disulfide bridge in the FP as a major factor for the high mechanical stability of the FP membrane anchor. We conclude, first, that the sequential binding of two short amphipathic helices allows the SARS-CoV-2 FP to insert quickly into the target membrane, before the virion is swept away after shedding the S1 domain connecting it to the host cell receptor. Second, we conclude that the double attachment and the conserved disulfide bridge establish the strong anchoring required for subsequent membrane fusion. Multiple distinct membrane-anchoring elements ensure high avidity and high mechanical strength of FP-membrane binding.

Dataset Information

Genetically Encoded Spy Peptide Fusion System to Detect Plasma Membrane-Localized Proteins In Vivo.

Publications

Genetically Encoded Spy Peptide Fusion System to Detect Plasma Membrane-Localized Proteins In Vivo.

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