Direct observation of the pH-dependent equilibrium between metarhodopsins I and II and the pH-independent interaction of metarhodopsin II with transducin C-terminal peptide.
Bovine rhodopsin contains 11-cis-retinal as a light-absorbing chromophore that binds to a lysine residue of the apoprotein opsin via a protonated Schiff base linkage. Light isomerizes 11-cis-retinal into the all-trans form, which eventually leads to the formation of an enzymatically active state, metarhodopsin II (MII). It is widely believed that MII forms a pH-dependent equilibrium with metarhodopsin I (MI), but direct evidence for this equilibrium has not been reported. Here, we confirmed this equilibrium by direct observation of the mutual conversions of MI and MII upon changing the pH of the MI/MII mixture. We also observed a reversible binding of the synthetic peptide constituting the C-terminal 11 amino acids of the transducin alpha-subunit to MII, which resulted in change of the amounts of MI and MII in the equilibrium. Interestingly, addition of the peptide did not induce a simple pK(a) shift but rather induced an increase of the MII fraction at high pH. These results indicate that in addition to the MII that is formed from MI in a pH-dependent manner there also exists another MII, which is in equilibrium with MI in a pH-independent manner and can bind to the peptide. Therefore, there is no need for proton uptake by the protein moiety of opsin for the binding to the peptide.
Sato
Keita
K
Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto 606-8502, Japan.
Morizumi
Takefumi
T
Yamashita
Takahiro
T
Shichida
Yoshinori
Y
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D012243:Rhodopsin / Q000737:chemistry* / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000378:metabolism; D015293:Transducin / Q000737:chemistry* / Q000378:metabolism
D010455:Peptides; C013110:metarhodopsins; D012243:Rhodopsin; D015293:Transducin