Linkage between the intramembrane H-bond network around aspartic acid 83 and the cytosolic environment of helix 8 in photoactivated rhodopsin.
Understanding the coupling between conformational changes in the intramembrane domain and at the membrane-exposed surface of the bovine photoreceptor rhodopsin, a prototypical G protein-coupled receptor (GPCR), is crucial for the elucidation of molecular mechanisms in GPCR activation. Here, we have combined Fourier transform infrared (FTIR) and fluorescence spectroscopy to address the coupling between conformational changes in the intramembrane region around the retinal and the environment of helix 8, a putative cytosolic surface switch region in class-I GPCRs. Using FTIR/fluorescence cross-correlation we show specifically that surface alterations monitored by emission changes of fluorescein bound to Cys316 in helix 8 of rhodopsin are highly correlated with (i) H-bonding to Asp83 proximal of the retinal Schiff base but not to Glu122 close to the beta-ionone and (ii) with a metarhodopsin II (MII)-specific 1643 cm(-1) IR absorption change, indicative of a partial loss of secondary structure in helix 8 upon MII formation. These correlations are disrupted by limited C-terminal proteolysis but are maintained upon binding of a transducin alpha-subunit (G(talpha))-derived peptide, which stabilizes the MII state. Our results suggest that additional C-terminal cytosolic loop contacts monitored by an amide II absorption at 1557 cm(-1) play a functionally crucial role in keeping helix 8 in the position in which its environment is strongly coupled to the retinal-binding site near the Schiff base. In the intramembrane region, this coupling is mediated by the H-bonding network that connects Asp83 to the NPxxY(x)F motif preceding helix 8.
Lehmann
Nicole
N
Institute of Radiation Physics, Biophysics Division, Forschungszentrum Dresden-Rossendorf, PF 510119, D-01314 Dresden, Germany.
Alexiev
Ulrike
U
Fahmy
Karim
K
D001224:Aspartic Acid / Q000737:chemistry*; D003553:Cystine / Q000737:chemistry; D003600:Cytosol / Q000737:chemistry*; D019793:Fluorescein / Q000737:chemistry; D007259:Infrared Rays; D007425:Intracellular Membranes / Q000737:chemistry*; D015394:Molecular Structure; D010777:Photochemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry*; D012243:Rhodopsin / Q000737:chemistry*; D013329:Structure-Activity Relationship
D043562:Receptors, G-Protein-Coupled; D001224:Aspartic Acid; D003553:Cystine; D012243:Rhodopsin; D019793:Fluorescein