Time-resolved fourier transform infrared study of structural changes in the last steps of the photocycles of Glu-204 and Leu-93 mutants of bacteriorhodopsin.
The last intermediate in the photocycle of the light-driven proton pump bacteriorhodopsin is the red-shifted O state. The structure and dynamics of the last step in the photocycle were characterized with time-resolved Fourier transform infrared spectroscopy of the mutants of Glu-204 and Leu-93, which accumulate this intermediate in much larger amounts than the wild type. The results show that E204Q and E204D give distorted all-trans-retinal chromophore like the O intermediate of the wild type. This is simply due to the perturbation of the proton acceptor function of Glu-204 in the O-to-BR transition in the Glu-204 mutants. The corresponding red-shifted intermediates of L93M, L93T, and L93S have a 13-cis chromophore like the N intermediate of the wild type, as reported from analysis of extracted retinal [Delaney, J. K., Schweiger, U., & Subramaniam, S. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 11120-11124]. In spite of their different chromophore structures from the O intermediate, the red-shifted intermediates are similar to the O intermediate but not to the N intermediate of the wild type with respect to structural changes in the peptide carbonyls. The structural changes around Asp-96 in the N intermediate are completely restored also in the red-shifted intermediates of the Leu-93 mutants like in the O intermediate. These results imply that the protein structural changes in the last step proceed regardless of thermal isomerization of the chromophore. Time-resolved Fourier transform infrared spectroscopy with the Glu-204 mutants suggests that the response of Asp-204 (Glu-204 in the wild type) to the protonation of Asp-85 during formation of the M intermediate, which results in proton release, is slow and may occur through structural changes.
Kandori
H
H
Department of Biophysics, Graduate School of Science, Kyoto University, Sakyo-ku, Japan.
Yamazaki
Y
Y
Hatanaka
M
M
Needleman
R
R
Brown
L S
LS
Richter
H T
HT
Lanyi
J K
JK
Maeda
A
A
D001436:Bacteriorhodopsins / Q000737:chemistry*; D005973:Glutamine; D007700:Kinetics; D007930:Leucine; D011487:Protein Conformation; D017550:Spectroscopy, Fourier Transform Infrared; D013329:Structure-Activity Relationship
D005973:Glutamine; D001436:Bacteriorhodopsins; D007930:Leucine