SITE-DIRECTED ISOTOPE LABELING AND ATR-FTIR DIFFERENCE SPECTROSCOPY OF BACTERIORHODOPSIN - THE PEPTIDE CARBONYL GROUP OF TYR-185 IS STRUCTURALLY ACTIVE DURING THE BR-]N-TRANSITION

The largest secondary structural change occurs in the bacteriorhodopsin (bR) photocycle during the M --> N transition. In this work site-directed isotope labeling (SDIL) and attenuated total reflection Fourier transform infrared (ATR-FTIR) difference spectroscopy were used to investigate this conformational change. L-Tyrosine containing a C-13 isotope at the carbonyl carbon was selectively incorporated at Tyr 57, Tyr 147, and Tyr 185 by SDIL. This involves the cell-free expression of bR in the presence of Escherichia coli suppressor tRNA(CUA)(Tyr) aminoacylated with L-[1-C-13]Tyr. ATR-FTIR difference spectroscopy reveals that of the 11 tyrosines, only the peptide carbonyl group of Tyr 185 undergoes a significant structural change during the bR --> N transition. Along with other spectroscopic evidence, this result suggests that the Tyr 185-Pro 186 region of the protein is structurally active and may function as a hinge which facilitates the tilt of the cytoplasmic portion of the F-helix in bacteriorhodopsin during the M --> N transition.