The role of Glu181 in the photoactivation of rhodopsin.
The visual pigment rhodopsin is a prototypical seven transmembrane helical G protein-coupled receptor. Photoisomerization of its protonated Schiff base (PSB) retinylidene chromophore initiates a progression of metastable intermediates. We studied the structural dynamics of receptor activation by FTIR spectroscopy of recombinant pigments. Formation of the active state, Meta II, is characterized by neutralization of the PSB and its counterion Glu113. We focused on testing the hypothesis of a PSB counterion switch from Glu113 to Glu181 during the transition of rhodopsin to the still inactive Meta I photointermediate. Our results, especially from studies of the E181Q mutant, support the view that both Glu113 and Glu181 are deprotonated, forming a complex counterion to the PSB in rhodopsin, and that the function of the primary counterion shifts from Glu113 to Glu181 during the transition to Meta I. The Meta I conformation in the E181Q mutant is less constrained compared with that of wild-type Meta I. In particular, the hydrogen bonded network linking transmembrane helices 1, 2, and 7, adopts a conformation that is already Meta II-like, while other parts of the receptor appear to be in a Meta I-like conformation similar to wild-type. We conclude that Glu181 is responsible, in part, for controlling the extraordinary high pK(a) of the chromophore PSB in the dark state, which very likely decreases upon transition to Meta I in a stepwise weakening of the interaction between PSB and its complex counterion during the course of receptor activation. A model for the specific role in coupling chromophore isomerization to protein conformational changes concomitant with receptor activation is presented.
Lüdeke
Steffen
S
Biophysics Group, Institut für Molekulare Medizin und Zellforschung, Albert-Ludwigs-Universität Freiburg, Hermann-Herder-Str. 9, D-79104 Freiburg, Germany.
Beck
Mareike
M
Yan
Elsa C Y
EC
Sakmar
Thomas P
TP
Siebert
Friedrich
F
Vogel
Reiner
R
D000818:Animals; D002417:Cattle; D018698:Glutamic Acid / Q000737:chemistry*; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D008027:Light; D008958:Models, Molecular; D011487:Protein Conformation; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D012545:Schiff Bases / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared
D012545:Schiff Bases; D018698:Glutamic Acid; D012243:Rhodopsin