Role of glutamate 134 of bovine rhodopsin in controlling the pKa and rate of deprotonation of the retinal schiff base

O. Kuwata, C. Yuan, T. G. Ebrey

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The tod visual pigment rhodopsin contains an 11-cis retinylidcne chromophore covalently attached to a lysine residue of the opsin via a Schiff base linkage. The Schiff base is protonated in the ground state of the pigment, but becomes deprotcmatcd after absorption of light to form the activated state of the pigment. Thus the light-controlled protonation/deprotonation of the Schiff base is one of the key physiological functions of the rhodopsin molecule. Glu113 in the third transmembrane α-helix of rhodopsin has been found to serve as a "countcrion" by electrostatically interacting with the Schiff base to stabilize its protonation and as the proton acceptor when the Schiff base deprotonates. However there are other residues that may interact either with the Schiff base or the countenon to further control the state of protonation of these two residues and/or the kinetics of protonation/deprotonation process. We are currently focusing on Glu134- which lies near the boundary between the third helix and intradiscal loop region, as a candidate for a such residue. We hypothesize that Giu134 would control the rate of deprotonation of the Schiff base by affecting the pKa of the counterion. In order to elucidate the role of Glu134 in the control mechanism, we have prepared rhodopsin mutants in which Glu134 is replaced with anoiher amino acid, such as glutamine to neutralize the negative charge, or aspartate to alter the distance of the charge at the 134 position from Glu113. A synthetic rhodopsin gene (courtesy of D. D. Oprianl was used lo construct genes with these mutations. Cultured COS-1 cells were transfected with the gene, and cell membranes containing the pigment isolated. Spectroscopic analyses of the mutant pigments, including pH dependent titration of the absorption spectra were conducted. We will discuss the results in the lieht of mechanism of interaction between Glu134 and the counterion.

Original languageEnglish (US)
Pages (from-to)S593
JournalInvestigative Ophthalmology and Visual Science
Issue number4
StatePublished - Dec 1 1997

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