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

作者：Hofmann Lukas; Alexander Nathan S; Sun Wenyu; Zhang Jianye; Orban Tivadar; Palczewski Krzysztof

来源：Biochemistry, 2017, 56(17): 2338-2348.

DOI：10.1021/acs.biochem.7b00165

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 N-32 and N-34 are 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.

• 出版日期2017-5-2