Transducin-dependent protonation of glutamic acid 134 in rhodopsin.
A highly conserved carboxylic acid residue in rhodopsin, Glu(134), modulates transducin (G(t)) interaction. It has been postulated that Glu(134) becomes protonated upon receptor activation. We studied the interaction between rhodopsin and G(t) using Fourier transform infrared (FTIR) difference spectroscopy combined with attenuated total reflection (ATR). Formation of the complex between G(t) 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 G(t), replacement of Glu(134) 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 Asp(83) and Glu(122) of rhodopsin. In addition, a negative absorption band at 1400 cm(-)(1) that is evoked by interaction of native metarhodopsin II (MII) with G(t) was not observed in the difference spectrum of the E134Q mutant. Thus, Glu(134) is ionized in the dark and exhibits a symmetrical COO(-) stretching vibration at 1400 cm(-)(1). Glu(134) becomes protonated in the G(t)-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 Glu(134) 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 G(t) binds. These results show directly that Glu(134) becomes protonated in MII upon G(t) binding and suggest that changes in receptor conformation affect lipid-protein interactions.
Fahmy
K
K
Sektion Biophysik, Institut fuer Molekulare Medizin und Zellforschung, Albert-Ludwigs-Universität, Albertstrasse 23, D-79104 Freiburg, Germany. fahmy@uni-freiburg.de
Sakmar
T P
TP
Siebert
F
F
D000818:Animals; D002417:Cattle; D018698:Glutamic Acid / Q000737:chemistry* / Q000378:metabolism*; D066298:In Vitro Techniques; D016297:Mutagenesis, Site-Directed; D010777:Photochemistry; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017550:Spectroscopy, Fourier Transform Infrared; D015293:Transducin / Q000378:metabolism*
D011522:Protons; D011994:Recombinant Proteins; D018698:Glutamic Acid; D012243:Rhodopsin; D015293:Transducin