TYROSINE STRUCTURAL-CHANGES DETECTED DURING THE PHOTOACTIVATION OF RHODOPSIN

We present the first Fourier transform infrared (FTIR) analysis of an isotope-labeled eukaryotic membrane protein. A combination of isotope labeling and FTIR difference spectroscopy was used to investigate the possible involvement of tyrosines in the photoactivation of rhodopsin (Rho), Rho --> MII difference spectra were obtained at 10 degrees C fo r unlabeled recombinant Rho and isotope-labeled L-[ring-H-2(4)]Tyr-Rho expressed in Spodoptera frugiperda cells grown on a stringent culture medium containing enriched L-[ring-H-2(4)]Tyr and isolated using a Hi s, tag, A comparison of these difference spectra revealed reproducible changes in bands that correspond to tyrosine and tyrosinate vibration al modes. A similar pattern of tyrosine/tyrosinate bands has also been observed in the bR --> M transition in bacteriorhodopsin, although th e sign of the bands is reversed. In bacteriorhodopsin, these bands wer e assigned to Tyr-185, which along with Pro-186 in the F-helix, may fo rm a hinge that facilitates alpha-helix movement.