biophys_publ_Transducin-Dependent Protonation of Glutamic Acid 134 in Rhodopsin
Fahmy, K.; Sakmar T.P., Siebert, F.
Transducin-Dependent Protonation of Glutamic Acid 134 in Rhodopsin
New York 10021 Received April 21, 2000 Revised Manuscript Received June 26, 2000
Abstract: A highly conserved carboxylic acid residue in rhodopsin, Glu134, modulates transducin (Gt) interaction. It has been postulated that Glu134 becomes protonated upon receptor activation. We studied the interaction between rhodopsin and Gt using Fourier transform infrared (FTIR) difference spectroscopy combined with attenuated total reflection (ATR). Formation of the complex between Gt and photoactivated rhodopsin reconstituted into phosphatidylcholine vesicles caused prominent infrared absorption increases at 1641, 1550, and 1517 cm-1. The rhodopsin mutant E134Q was also studied. When measured in the presence of Gt, replacement of Glu134 by glutamine abolished the low-frequency part of a broad absorption band at 1735 cm-1 that is normally superimposed on the light-induced absorption changes of Asp83 ad Glu122 of rhodopsin. In addition, a negative absorption band at 1400 cm-1 that is evoked by interaction of native metarhodopsin II (MII) with Gt was not observed in the difference spectrum of the E134Q mutant. Thus, Glu134 is ionized in the dark and exhibits a symmetrical COO- stretching vibration at 1400 cm-1. Glu134 becomes protonated in the Gt-MII complex and displays a C=O stretching mode near 1730 cm-1. The E134Q mutation also affects absorption changes attributable to lipids, suggesting that the protonation of Glu134 is linked to transfer of the carboxylic acid side chain from a polar to a nonpolar environment by becoming exposed to the lipid phase when Gt binds. These results show directly that Glu134 becomes protonated in MII upon Gt binding and suggest that changes in receptor conformation affect lipid-protein interactions.