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 N32 and N34 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.
Subject(s)
Cone Opsins/chemistry , Receptors, G-Protein-Coupled/chemistry , Rod Opsins/chemistry , Amino Acid Motifs , Amino Acid Substitution , Asparagine/metabolism , Binding Sites , Computational Biology , Cone Opsins/genetics , Cone Opsins/metabolism , Cone Opsins/radiation effects , Conserved Sequence , Deuterium Exchange Measurement , Glycosylation , Humans , Ligands , Light , Point Mutation , Proline/chemistry , Protein Conformation , Protein Refolding/radiation effects , Protein Structure, Tertiary , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Receptors, G-Protein-Coupled/radiation effects , Recombinant Proteins , Rod Opsins/genetics , Rod Opsins/metabolism , Rod Opsins/radiation effects , Spectrometry, Mass, Electrospray Ionization , Tandem Mass SpectrometryABSTRACT
Photosynthetic cyanobacteria make important contributions to global carbon and nitrogen budgets. A protein known as the orange carotenoid protein (OCP) protects the photosynthetic apparatus from damage by dissipating excess energy absorbed by the phycobilisome, the major light-harvesting complex in many cyanobacteria. OCP binds one carotenoid pigment, but the color of this pigment depends on conditions. It is orange in the dark and red when exposed to light. We modified the orange and red forms of OCP by using isotopically coded cross-linking agents and then analyzed the structural features by using liquid chromatography and tandem mass spectrometry. Unequivocal cross-linking pairs uniquely detected in red OCP indicate that, upon photoactivation, the OCP N-terminal domain (NTD) and C-terminal domain (CTD) reorient relative to each other. Our data also indicate that the intrinsically unstructured loop connecting the NTD and CTD not only is involved in the interaction between the two domains in orange OCP but also, together with the N-terminal extension, provides a structural buffer system facilitating an intramolecular breathing motion of the OCP, thus helping conversion back and forth from the orange to red form during the OCP photocycle. These results have important implications for understanding the molecular mechanism of action of cyanobacterial photoprotection.
Subject(s)
Bacterial Proteins/chemistry , Carotenoids/chemistry , Models, Molecular , Synechocystis/metabolism , Bacterial Proteins/metabolism , Carotenoids/metabolism , Carotenoids/radiation effects , Chromatography, High Pressure Liquid , Cross-Linking Reagents/chemistry , Dimerization , Ligands , Light , Molecular Weight , Peptide Mapping , Photochemical Processes , Protein Refolding/radiation effects , Protein Structure, Tertiary/radiation effects , Tandem Mass SpectrometryABSTRACT
Aggregation of lysozyme in an acidic solution generates inactive amyloid-like fibrils, with a broad infrared peak appearing at 1,610-1,630 cm(-1), characteristic of a ß-sheet rich structure. We report here that spontaneous refolding of these fibrils in water could be promoted by mid-infrared free-electron laser (mid-IR FEL) irradiation targeting the amide bands. The Fourier transform infrared spectrum of the fibrils reflected a ß-sheet content that was as low as that of the native structure, following FEL irradiation at 1,620 cm(-1) (amide I band); both transmission-electron microscopy imaging and Congo Red assay results also demonstrated a reduced fibril structure, and the enzymatic activity of lysozyme fibrils recovered to 70-90 % of the native form. Both irradiations at 1,535 cm(-1)(amide II band) and 1,240 cm(-1) (amide III band) were also more effective for the refolding of the fibrils than mere heating in the absence of FEL. On the contrary, either irradiation at 1,100 or 2,000 cm(-1) afforded only about 60 % recovery of lysozyme activity. These results indicate that the specific FEL irradiation tuned to amide bands is efficient in refolding of lysozyme fibrils into native form.
