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 13C 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(CUATyr) aminoacylated with L-[1-13C]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.
Ludlam
C F
CF
Physics Department, Boston University, Massachusetts 02215.
Sonar
S
S
Lee
C P
CP
Coleman
M
M
Herzfeld
J
J
RajBhandary
U L
UL
Rothschild
K J
KJ
D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000528:radiation effects; D002247:Carbon Isotopes; D007553:Isotope Labeling; D010777:Photochemistry; D017433:Protein Structure, Secondary / Q000528:radiation effects*; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods; D014443:Tyrosine / Q000737:chemistry
D002247:Carbon Isotopes; D014443:Tyrosine; D001436:Bacteriorhodopsins