PROTEIN STRUCTURAL-CHANGES IN BACTERIORHODOPSIN UPON PHOTOISOMERIZATION AS REVEALED BY POLARIZED FTIR SPECTROSCOPY

We used polarized Fourier transform infrared (FTIR) spectroscopy to in vestigate the structural change of bacteriorhodopsin (BR) upon photois omerization of the retinal chromophore. By measuring the difference sp ectra between the K-intermediate and BR in the whole mid-infrared regi on (700-4000 cm(-1)) at 77 K, complete vibrational information was obt ained on how the protein responds to the displacement of the chromopho re. In particular, changes in O-H and N-H stretching vibrations, which directly probe the hydrogen bonding strength, have provided not only the relevant frequencies but also their angles to the membrane normal. Structural perturbation of the peptide backbone appears in the 3270-3 320 cm(-1) (peptide N-H stretch) and the 1650-1670 cm(-1) (peptide C=O stretch) regions. These peptide bands are insensitive to H-D exchange , and the dipole moments of the N-H and C=O stretches are parallel to the membrane normal. In contrast, several bands are downshifted upon D 2O substitution, indicating that O-H or N-H groups that participate in a hydrogen bonding network near the chromophore change upon cis-trans isomerization.