The molecular origin of the inhibition of transducin activation in rhodopsin lacking the 9-methyl group of the retinal chromophore: A UV-Vis and FTIRspectroscopic study

The formation of the active rhodopsin state metarhodopsin Il (MII) is belie ved to be partially governed by specific steric constraints imposed onto th e protein by the 9-methyl group of the retinal chromophore. We studied the pl properties of the synthetic pigment 9-demethyl rhodopsin (Bdm-Rho), cons isting of the rhodopsin apoprotein regenerated with synthetic retinal lacki ng the 9-methyl group, by UV-vis and Fourier transform infrared difference spectroscopy. Low activation rates of the visual G-protein transducin by th e modified pigment reported in previous studies are shown to not be caused by the reduced activity of its MII state, but to be due to a dramatic equil ibrium shift from MII to its immediate precursor, MI. The MII state of 9dm- Rho displays only a partial deprotonation of the retinal Schiff base, leadi ng to the formation of two MII subspecies absorbing at 380 and 470 nm, both of which seem to be involved in transducin activation. The rate of MII for mation is slowed by 2 orders of magnitude: compared to rhodopsin. The dark state and the MI state of 9dm-Rho are distinctly different from their respe ctive states in the native pigment, pointing to a more relaxed fit of the r etinal chromophore in its binding pocket. The shifted equilibrium between M I and MII is therefore discussed in terms of an increased entropy of the 9d m-Rho MI state due to changed steric interactions.