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Title: An engineered opsin monomer scrambles phospholipids

Abstract

The G protein-coupled receptor opsin is a phospholipid scramblase that facilitates rapid transbilayer phospholipid exchange in liposomes. The mechanism by which opsin scrambles lipids is unknown. It has been proposed that lipid translocation may occur at protein-protein interfaces of opsin dimers. To test this possibility, we rationally engineered QUAD opsin by tryptophan substitution of four lipid-facing residues in transmembrane helix 4 (TM4) that is known to be important for dimerization. Atomistic molecular dynamics simulations of wild type and QUAD opsins combined with continuum modeling revealed that the tryptophan substitutions lower the energetically unfavorable residual hydrophobic mismatch between TM4 and the membrane, reducing the drive of QUAD opsin to dimerize. We purified thermostable wild type and QUAD opsins, with or without a SNAP tag for fluorescence labeling. Single molecule fluorescence measurements of purified SNAP-tagged constructs revealed that both proteins are monomers. Fluorescence-based activity assays indicated that QUAD opsin is a fully functional scramblase. However, unlike wild type opsin which dimerizes en route to insertion into phospholipid vesicles, QUAD opsin reconstitutes as a monomer. We conclude that an engineered opsin monomer can scramble phospholipids, and that the lipid-exposed face of TM4 is unlikely to contribute to transbilayer phospholipid exchange.

Authors:
 [1];  [1];  [1];  [1];  [2]; ORCiD logo [1]
  1. Weill Cornell Medical College, New York, NY (United States). Dept. of Biochemistry
  2. Weill Cornell Medical College, New York, NY (United States). Dept. of Physiology and Biophysics. Inst. for Computational Biomedicine
Publication Date:
Research Org.:
Weill Cornell Medical College, New York, NY (United States)
Sponsoring Org.:
USDOE Office of Science (SC); National Inst. of Health (NIH) (United States); Velux Stiftung (Switzerland); Austrian Science Foundation (FWF)
OSTI Identifier:
1490796
Grant/Contract Number:  
AC02-05CH11231; EY024207; EY027969; J3686-B21
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Scientific Reports
Additional Journal Information:
Journal Volume: 7; Journal ID: ISSN 2045-2322
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; membranes; permeation and transport; phospholipids; protein function predictions; transporters

Citation Formats

Pandey, Kalpana, Ploier, Birgit, Goren, Michael A., Levitz, Joshua, Khelashvili, George, and Menon, Anant K. An engineered opsin monomer scrambles phospholipids. United States: N. p., 2017. Web. doi:10.1038/s41598-017-16842-z.
Pandey, Kalpana, Ploier, Birgit, Goren, Michael A., Levitz, Joshua, Khelashvili, George, & Menon, Anant K. An engineered opsin monomer scrambles phospholipids. United States. doi:10.1038/s41598-017-16842-z.
Pandey, Kalpana, Ploier, Birgit, Goren, Michael A., Levitz, Joshua, Khelashvili, George, and Menon, Anant K. Fri . "An engineered opsin monomer scrambles phospholipids". United States. doi:10.1038/s41598-017-16842-z. https://www.osti.gov/servlets/purl/1490796.
@article{osti_1490796,
title = {An engineered opsin monomer scrambles phospholipids},
author = {Pandey, Kalpana and Ploier, Birgit and Goren, Michael A. and Levitz, Joshua and Khelashvili, George and Menon, Anant K.},
abstractNote = {The G protein-coupled receptor opsin is a phospholipid scramblase that facilitates rapid transbilayer phospholipid exchange in liposomes. The mechanism by which opsin scrambles lipids is unknown. It has been proposed that lipid translocation may occur at protein-protein interfaces of opsin dimers. To test this possibility, we rationally engineered QUAD opsin by tryptophan substitution of four lipid-facing residues in transmembrane helix 4 (TM4) that is known to be important for dimerization. Atomistic molecular dynamics simulations of wild type and QUAD opsins combined with continuum modeling revealed that the tryptophan substitutions lower the energetically unfavorable residual hydrophobic mismatch between TM4 and the membrane, reducing the drive of QUAD opsin to dimerize. We purified thermostable wild type and QUAD opsins, with or without a SNAP tag for fluorescence labeling. Single molecule fluorescence measurements of purified SNAP-tagged constructs revealed that both proteins are monomers. Fluorescence-based activity assays indicated that QUAD opsin is a fully functional scramblase. However, unlike wild type opsin which dimerizes en route to insertion into phospholipid vesicles, QUAD opsin reconstitutes as a monomer. We conclude that an engineered opsin monomer can scramble phospholipids, and that the lipid-exposed face of TM4 is unlikely to contribute to transbilayer phospholipid exchange.},
doi = {10.1038/s41598-017-16842-z},
journal = {Scientific Reports},
issn = {2045-2322},
number = ,
volume = 7,
place = {United States},
year = {2017},
month = {12}
}

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