An engineered opsin monomer scrambles phospholipids Academic Article uri icon

Overview

MeSH Major

  • Klebsiella pneumoniae
  • beta-Lactamases

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.

publication date

  • December 2017

Research

keywords

  • Academic Article

Identity

Language

  • eng

PubMed Central ID

  • PMC5711885

Digital Object Identifier (DOI)

  • 10.1038/s41598-017-16842-z

PubMed ID

  • 29196630

Additional Document Info

start page

  • 16741

volume

  • 7

number

  • 1