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

Abstract

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.

Rod Visual Pigment Optimizes Active State to Achieve Efficient G Protein Activation as Compared with Cone Visual Pigments

“…6D). Using this b0 spectrum, the fraction of Meta-II in Meta-I/Meta-II equilibrium (F Meta-II ) was estimated as described previously (36,37) (Table 3). F Meta-II in nanodiscs was 51% at 20°C, which was consistent with that in ROSs (57%), whereas F Meta-II in DM-solubilized bvRh was 100%, which was considerably higher than F Meta-II in nanodiscs and ROSs.…”

“…b0 spectra, which were difference spectra in the equilibrium state, of bvRh in nanodiscs and ROSs and DM suspension are shown in D (solid lines for bvRh in nanodiscs at 0 -37°C and dashed lines for bvRh in ROSs or DM suspension at 20°C). These spectra were fitted by Meta-I and Meta-II model spectra as described previously (36,37) to estimate F Meta-II (Table 3). Diff.…”

Phospholipid Bicelles Improve the Conformational Stability of Rhodopsin Mutants Associated with Retinitis Pigmentosa

“…Some of these mutations can cause structural instability and misfolding 42 . The stability of visual photoreceptors, and other GPCRs, has been extensively studied, and several experimental factors, like temperature, pH, salts, detergents, and lipids have been shown to affect the stability and function of these receptors 8,22,28,[43][44][45][46] . In spite of this, only limited information is available concerning the molecular causes of the structural instability of mutations in Rho associated with RP.…”

Complexity of Bovine Rhodopsin Activation Revealed at Low Temperature and Alkaline pH

“…The scheme presented here, however, is consistent with EPR studies describing the helix-6 movement involved in activation, 31 and G-protein peptide mimic binding studies. 41 Many structural studies reveal complex helix movements involving the rearrangements of H-bonding in the transmembrane region of the protein, and several states of the chromophore relaxation during activation. [21][22][23][24][25][26][27]42 Since the structural studies that reveal those motions are carried out by trapping intermediates at low temperatures and only some of the intermediates are likely to be trapped at low temperature, it is not possible to identify specific structures with specific intermediates observed here.…”