FTIR spectroscopy reveals microscopic structural changes of the protein around the rhodopsin chromophore upon photoisomerization.
Fourier transform infrared spectroscopy was used to investigate the local structure around the chromophore of rhodopsin and its change upon photoisomerization. A hydrated film of bovine rod outer segments was cooled at 80 K, and difference infrared spectra were obtained between bathorhodopsin and rhodopsin or between bathorhodopsin and isorhodopsin under suitable irradiation conditions. The spectra in a higher-frequency region (4000-1800 cm-1) revealed protein structural change by probing the O-H, N-H, and S-H stretching vibrational modes. The structural change of bound water molecules occurred upon formation of bathorhodopsin, where three water O-H increased the strength of their H-bonding. The water structure is identical in rhodopsin and isorhodopsin. These results suggest that the protein in the close proximity of the Schiff base of the chromophore is perturbed upon photoisomerization and causes rearrangement of the water molecules in bathorhodopsin. Upon the isomerization, the 3463 cm-1 band of the 11-cis form (rhodopsin) shifts to 3487 cm-1 for the all-trans form (bathorhodopsin) or to 3481 cm-1 for the 9-cis form (isorhodopsin). An N-H bond, possibly of an indole of tryptophan residue, is responsible for these bands. It is present in a hydrophobic environment around the beta-ionone ring and/or polyene chain of the retinal, and changes its geometrical alignment depending on the isomeric state. It is the only band distinct in frequency between rhodopsin and isorhodopsin in the high-frequency region, suggesting that the specific interaction between the N-H and the chromophore contributes to the more efficient isomerization in rhodopsin than isorhodopsin. The stretching vibrations of the water O-H, cysteine S-H, and amide N-H of the peptide backbone decrease in frequency upon formation of bathorhodopsin, indicating that H-bonding around the chromophore becomes stronger in bathorhodopsin. This shows that at least a part of the energy absorbed in the chromophore is already transferred to the protein in bathorhodopsin by strengthened H-bonding. The chromophore-protein interaction as a suitable reaction field in rhodopsin is discussed on the basis of these observations.
Kandori
H
H
Department of Biophysics, Graduate School of Science, Kyoto University, Japan.
Maeda
A
A
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003080:Cold Temperature; D005136:Eye Proteins / Q000737:chemistry*; D007536:Isomerism; D008969:Molecular Sequence Data; D010777:Photochemistry; D011487:Protein Conformation; D012243:Rhodopsin / Q000737:chemistry*; D012374:Rod Cell Outer Segment / Q000737:chemistry; D017550:Spectroscopy, Fourier Transform Infrared
D005136:Eye Proteins; D012243:Rhodopsin