title: Hydrogen/Deuterium Exchange Mass Spectrometry of Human Green Opsin Reveals a Conserved Pro-Pro Motif in Extracellular Loop 2 of Monostable Visual G Protein-Coupled Receptors.
abstract: Opsins comprise the protein component of light sensitive G protein-coupled receptors (GPCRs) in the retina of the eye that are responsible for the transduction of light into a biochemical signal. Here, we used hydrogen/deuterium (H/D) exchange coupled with mass spectrometry to map conformational changes in green cone opsin upon light activation. We then compared these findings with those reported for rhodopsin. The extent of H/D exchange in green cone opsin was greater than in rhodopsin in the dark and bleached states, suggesting a higher structural heterogeneity for green cone opsin. Further analysis revealed that green cone opsin exists as a dimer in both dark (inactive) and bleached (active) states, and that the predicted glycosylation sites at N32and N34are indeed glycosylated. Comparison of deuterium uptake between inactive and active states of green cone opsin also disclosed a reduced solvent accessibility of the extracellular N-terminal region and an increased accessibility of the chromophore binding site. Increased H/D exchange at the extracellular side of transmembrane helix four (TM4) combined with an analysis of sequence alignments revealed a conserved Pro-Pro motif in extracellular loop 2 (EL2) of monostable visual GPCRs. These data present new insights into the locus of chromophore release at the extracellular side of TM4 and TM5 and provide a foundation for future functional evaluation.
authors.affiliation: Department of Pharmacology and Cleveland Center for Membrane and Structural Biology, School of Medicine, Case Western Reserve University , 10900 Euclid Avenue, Cleveland, Ohio 44106, United States.
authors.forename: Lukas
authors.initials: L
authors.lastname: Hofmann
authors.affiliation: Department of Pharmacology and Cleveland Center for Membrane and Structural Biology, School of Medicine, Case Western Reserve University , 10900 Euclid Avenue, Cleveland, Ohio 44106, United States.
authors.forename: Nathan S
authors.initials: NS
authors.lastname: Alexander
authors.affiliation: Department of Pharmacology and Cleveland Center for Membrane and Structural Biology, School of Medicine, Case Western Reserve University , 10900 Euclid Avenue, Cleveland, Ohio 44106, United States.
authors.forename: Wenyu
authors.initials: W
authors.lastname: Sun
authors.affiliation: Department of Pharmacology and Cleveland Center for Membrane and Structural Biology, School of Medicine, Case Western Reserve University , 10900 Euclid Avenue, Cleveland, Ohio 44106, United States.
authors.forename: Jianye
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Department of Pharmacology and Cleveland Center for Membrane and Structural Biology, School of Medicine, Case Western Reserve University , 10900 Euclid Avenue, Cleveland, Ohio 44106, United States.
authors.forename: Tivadar
authors.initials: T
authors.lastname: Orban
authors.affiliation: Department of Pharmacology and Cleveland Center for Membrane and Structural Biology, School of Medicine, Case Western Reserve University , 10900 Euclid Avenue, Cleveland, Ohio 44106, United States.
authors.forename: Krzysztof
authors.identifier: 0000-0002-0788-545X
authors.initials: K
authors.lastname: Palczewski
mesh_terms: D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D001216:Asparagine / Q000378:metabolism; D001665:Binding Sites; D019295:Computational Biology; D055358:Cone Opsins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D017124:Conserved Sequence; D041961:Deuterium Exchange Measurement; D006031:Glycosylation; D006801:Humans; D008024:Ligands; D008027:Light; D017354:Point Mutation; D011392:Proline / Q000737:chemistry; D011487:Protein Conformation; D058849:Protein Refolding / Q000528:radiation effects; D017434:Protein Structure, Tertiary; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D011994:Recombinant Proteins; D017299:Rod Opsins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D021241:Spectrometry, Mass, Electrospray Ionization; D053719:Tandem Mass Spectrometry
chemical_list: D055358:Cone Opsins; D008024:Ligands; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; D017299:Rod Opsins; C095854:middle-wavelength opsin; D001216:Asparagine; D011392:Proline