Characterization of the conformational change in the M1 and M2 substates of bacteriorhodopsin by the combined use of visible and infrared spectroscopy.
A combination of visible and Fourier transform infrared (FTIR) spectroscopies is used to characterize the formation of the M1 and M2 substates of the bacteriorhodopsin photocycle in glucose-embedded, hydrated thin films. Difference FTIR bands in the amide I region verify the previously reported existence of a significant peptide backbone conformational change in the transition from M1 to M2. The visible absorption spectra demonstrate that contamination of the M-intermediate samples by L, N, or other non-M species should contribute negligibly to the observed changes in the amide I region, and this conclusion is supported by comparison of specific carboxyl group peaks with corresponding bands in published L and N FTIR difference spectra. Based upon spectroscopic results, an extension of the C-T Model (Fodor, S., Ames, J., Gebhard, R., van den Berg, E., Stoeckenius, W., Lugtenberg, J., and Mathies, R. (1988) Biochemistry 27, 7097-7101) is presented. The results of this work suggest that protein structural changes should be clearly visible in M-bR, difference Fourier density maps and that these structural changes may in turn elucidate how bacteriorhodopsin actively pumps ions across the purple membrane of Halobacterium halobium.
Perkins
G A
GA
Graduate Group in Biophysics, Lawrence Berkeley Laboratory, University of California, Berkeley 94720.
Liu
E
E
Burkard
F
F
Berry
E A
EA
Glaeser
R M
RM
D001436:Bacteriorhodopsins / Q000737:chemistry / Q000528:radiation effects / Q000648:ultrastructure*; D003460:Crystallization; D005583:Fourier Analysis; D019617:Halobacterium salinarum / Q000737:chemistry; D007700:Kinetics; D010777:Photochemistry; D011487:Protein Conformation; D013055:Spectrophotometry, Infrared; D013057:Spectrum Analysis; D013696:Temperature
D001436:Bacteriorhodopsins