The molecular origin of the inhibition of transducin activation in rhodopsin lacking the 9-methyl group of the retinal chromophore: a UV-Vis and FTIR spectroscopic study.
The formation of the active rhodopsin state metarhodopsin II (MII) is believed to be partially governed by specific steric constraints imposed onto the protein by the 9-methyl group of the retinal chromophore. We studied the properties of the synthetic pigment 9-demethyl rhodopsin (9dm-Rho), consisting of the rhodopsin apoprotein regenerated with synthetic retinal lacking the 9-methyl group, by UV-vis and Fourier transform infrared difference spectroscopy. Low activation rates of the visual G-protein transducin by the modified pigment reported in previous studies are shown to not be caused by the reduced activity of its MII state, but to be due to a dramatic equilibrium shift from MII to its immediate precursor, MI. The MII state of 9dm-Rho displays only a partial deprotonation of the retinal Schiff base, leading to the formation of two MII subspecies absorbing at 380 and 470 nm, both of which seem to be involved in transducin activation. The rate of MII formation is slowed by 2 orders of magnitude compared to rhodopsin. The dark state and the MI state of 9dm-Rho are distinctly different from their respective states in the native pigment, pointing to a more relaxed fit of the retinal chromophore in its binding pocket. The shifted equilibrium between MI and MII is therefore discussed in terms of an increased entropy of the 9dm-Rho MI state due to changed steric interactions.
Vogel
R
R
Sektion Biophysik, Institut für Molekulare Medizin und Zellforschung, Albert-Ludwig-Universität Freiburg, Albertstrasse 23, D-79104 Freiburg, Germany.
Fan
G B
GB
Sheves
M
M
Siebert
F
F
D000818:Animals; D000838:Anions; D002417:Cattle; D019277:Entropy; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D010782:Photolysis; D011487:Protein Conformation; D012172:Retinaldehyde / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D012243:Rhodopsin / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D017550:Spectroscopy, Fourier Transform Infrared; D013329:Structure-Activity Relationship; D015293:Transducin / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism
C110176:9-demethylretinal; D000838:Anions; C013110:metarhodopsins; D012243:Rhodopsin; D015293:Transducin; D012172:Retinaldehyde