Protein Structural Changes in Bacteriorhodopsin upon Photoisomerization As Revealed by Polarized FTIR Spectroscopy

Abstract

We used polarized Fourier transform infrared (FTIR) spectroscopy to investigate the structural change of bacteriorhodopsin (BR) upon photoisomerization of the retinal chromophore. By measuring the difference spectra between the K-intermediate and BR in the whole mid-infrared region (700−4000 cm-1) at 77 K, complete vibrational information was obtained on how the protein responds to the displacement of the chromophore. 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 3270−3320 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 D2O substitution, indicating that O−H or N−H groups that participate in a hydrogen bonding network near the chromophore change upon cis−trans isomerization.

Local and distant protein structural changes on photoisomerization of the retinal in bacteriorhodopsin

“…The dotted lines in Fig. 2 (23). The latter value is at a lower frequency than the values of neat secondary alcohol (3,340-3,355 cm Ϫ1 ), indicating strong interaction with a hydrogen-bond acceptor.…”

“…Therefore, it is unexpected for a conformation change to be transmitted over 11 Å on formation of the K intermediate. However, the selective and efficient nature of the photoisomerization in BR has been attributed to the surrounding protein (23). We therefore suggest that the specific protein environment involving Thr-17 may work as the specific reaction field of photoisomerization.…”

“…Polarized FTIR spectroscopy was applied as described (23,25). A 120-l aliquot of the sample in 2 mM phosphate buffer (pH 7.0) was dried on a BaF 2 window with diameter of 18 mm.…”

“…These results suggest that nuclear motions of the chromophore and surrounding protein would be minimal at the earliest stages. According to our low-temperature polarized Fourier-transform IR (FTIR) spectroscopy, however, a number of bands were observed from the protein that changed between the BR and the K intermediate (23). Because the formation and the relaxation of the K intermediate are coupled to structural rearrangement for efficient photoisomerization and proton translocation, respectively, a detailed analysis is required.…”

Tight Asp-85–Thr-89 association during the pump switch of bacteriorhodopsin

“…) region provided direct assessment of hydrogen bonds by probing OOH and NOH stretching vibrations (25). Furthermore, combined studies of isotopelabeled and mutant BR can lead to identification of particular vibrational bands.…”