Methionine changes in bacteriorhodopsin detected by FTIR and cell-free selenomethionine substitution.
Bacteriorhodopsin (BR) is an integral membrane protein, which functions as a light-driven proton pump in Halobacterium salinarum. We report evidence that one or more methionine residues undergo a structural change during the BR-->M portion of the BR photocycle. Selenomethionine was incorporated into BR using a cell-free protein translation system containing an amino acid mixture with selenomethionine substituted for methionine. BR-->M FTIR difference spectra recorded for unlabeled and selenomethionine-labeled cell-free expressed BR closely resemble the spectra of in vivo expressed BR. However, reproducible changes occur in two regions near 1,284 and 900 cm(-1) due to selenomethionine incorporation. Isotope labeled tyrosine was also co-incorporated with selenomethionine in order to confirm these assignments. Based on recent x-ray crystallographic studies, likely methionines which give rise to the FTIR difference bands are Met-118 and Met-145, which are located inside the retinal binding pocket and in a position to constrain the motion of retinal during photoisomerization. The assignment of methionine bands in the FTIR difference spectrum of BR provides a means to study methionine-chromophore interaction under physiological conditions. More generally, combining cell-free incorporations of selenomethionine into proteins with FTIR difference spectroscopy provides a useful method for investigating the role of methionines in protein structure and function.
Bergo
Vladislav
V
Physics Deptartment, Boston University, Boston, Massachusetts 02215, USA.
Mamaev
Sergey
S
Olejnik
Jerzy
J
Rothschild
Kenneth J
KJ
D019943:Amino Acid Substitution; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000302:isolation & purification; D002474:Cell-Free System / Q000737:chemistry*; D008715:Methionine / Q000737:chemistry*; D011487:Protein Conformation; D017510:Protein Folding; D012645:Selenomethionine / Q000737:chemistry*; D017550:Spectroscopy, Fourier Transform Infrared / Q000379:methods*; D013194:Staining and Labeling / Q000379:methods
D001436:Bacteriorhodopsins; D012645:Selenomethionine; D008715:Methionine