biophys_publ_Photoactivated state of rhodopsin and how it can form

Fahmy,  K., Siebert,  F., Sakmar, T.P.
Photoactivated state of rhodopsin and how it can form
Biophys Chem. 1995 Sep-Oct;56(1-2):171-81. Review

Abstract: A variety of spectroscopic and biochemical studies of the photoreceptor rhodopsin have revealed conformation changes which occur upon its photoactivation. Assignment of these molecular alterations to specific regions in the receptor has been attempted by studying native opsin regenerated with synthetic retinal analogs or recombinant opsins regenerated with 11-cis retinal. We propose a model for the photoactivation mechanism which defines 'off' and 'on' states for individual molecular groups. These groups have been identified to undergo structural alterations during photoactivation. Analysis of mutant pigments in which specific groups are locked into their respective 'on' or 'off' states provides a framework to identify determinants of the active conformation as well as the minimal number of intramolecular transitions to switch to this conformation. The simple model proposed for the active-state of rhodopsin can be compared to structural models of its ground-state to localize chromophore-protein interactions that may be important in the photoactivation mechanism. A mutant rhodopsin photoproduct with a protonated Schiff base displays an active-state conformation: a Fourier-transform infrared spectroscopy study. In the rhodopsin mutant E113A/A117E the position of the protonated Schiff base counterion, Glu113, is moved by one helix turn from position 113 to 117. The photoreaction of this mutant pigment was studied by Fourier-transform infrared (FTIR) difference spectroscopy. At acidic pH, formation of a 474-nm absorbing photoproduct previously characterized biochemically as a species that activates transducin caused infrared absorption changes typical of metarhodopsin II (MII) formation in native rhodopsin. Specific spectral alterations revealed a localized perturbation near the protonated Schiff base in the dark state. In addition, an infrared band assigned to the C = O stretching vibration of Glu113 in MII of rhodopsin was abolished in the mutant. Absorption changes caused by Asp83 and Glu122 C = O stretching vibrations characteristic of rhodopsin MII formation were not affected. At alkaline pH, mutant E113A/A117E formed predominantly a 382-nm absorbing photoproduct. It displayed infrared-difference absorption bands significantly different from those of native MII over a large spectral range. These results support the conclusion that the 474-nm photoproduct of mutant E113A/A117E, despite a protonated Schiff base linkage, displays a predominantly MII-like conformation capable of catalyzing guanine-nucleotide exchange by transducin.