Fourier transform infrared difference spectroscopy of bacteriorhodopsin and its photoproducts regenerated with deuterated tyrosine.
Fourier transform infrared (FTIR) difference spectroscopy has been used to detect the vibrational modes due to tyrosine residues in the protein that change in position or intensity between light-adapted bacteriorhodopsin (LA) and other species, namely, the K and M intermediates and dark-adapted bacteriorhodopsin (DA). To aid in the identification of the bands that change in these various species, the FTIR spectra of the free amino acids Tyr-d0, Tyr-d2 (2H at positions ortho to OH), and Tyr-d4 (2H at positions ortho and meta to OH) were measured in H2O and D2O at low and high pH. The characteristic frequencies of the Tyr species obtained in this manner were then used to identify the changes in protonation state of the tyrosine residues in the various bacteriorhodopsin species. The two diagnostically most useful bands were the approximately 1480-cm-1 band of Tyr(OH)-d2 and the approximately 1277-cm-1 band of Tyr(O-)-d0. Mainly by observing the appearance or disappearance of these bands in the difference spectra of pigments incorporating the tyrosine isotopes, it was possible to identify the following: in LA, one tyrosine and one tyrosinate; in the K intermediate, two tyrosines; in the M intermediate, one tyrosine and one tyrosinate; and in DA, two tyrosines. Since these residues were observed in the difference spectra K/LA, M/LA, and DA/LA, they represent the tyrosine or tyrosinate groups that most likely undergo changes in protonation state due to the conversions. These changes are most likely linked to the proton translocation process of bacteriorhodopsin.
D001436:Bacteriorhodopsins / Q000031:analogs & derivatives* / Q000378:metabolism*; D003903:Deuterium; D005583:Fourier Analysis; D006217:Halobacterium / Q000378:metabolism; D010782:Photolysis; D011487:Protein Conformation; D013055:Spectrophotometry, Infrared / Q000379:methods; D014443:Tyrosine
D014443:Tyrosine; D001436:Bacteriorhodopsins; D003903:Deuterium