Methionine Changes in Bacteriorhodopsin Detected by FTIR and Cell-Free Selenomethionine Substitution

Abstract

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.

Photoactivation Perturbs the Membrane-embedded Contacts between Sensory Rhodopsin II and Its Transducer

“…This time range is associated predominantly with accumulation of the M intermediate (24), which is a signaling state for the transducer activation (29). Several prominent bands appear at 1163 (Ϫ), 1200 (Ϫ), 1240 (Ϫ), 1544 (Ϫ), and 1567 (ϩ) cm Ϫ1 in the spectra of wild-type SRII-HtrII complex (top trace), which have been previously assigned to the major vibrations of the retinylidene chromophore in the all-trans (negative bands) and 13-cis (positive bands) states as the receptor undergoes a transition from the dark state to the M intermediate (24,30). The presence of M is also evident from the strong positive band near 1764 cm Ϫ1 due to protonation of the Schiff base counterion Asp 75 and prominent bands in the amide I region at 1644 (ϩ) and 1664 (Ϫ) cm Ϫ1 (24,25).…”

Conformational changes in the archaerhodopsin-3 proton pump: detection of conserved strongly hydrogen bonded water networks

“…Only the frequency of bands discussed in this paper are labeled in this and subsequent figures a slight upshift measured by resonance Raman spectroscopy (RRS data not shown). An empirical inverse relationship between the visible absorption maximum and the ethylenic stretching frequency has been established for rhodopsins [33] and microbial rhodopsins (see for example [21,26,34]), which indicates that such an upshift might correspond to a few nanometer blue-shift of the λ max of AR3 relative to BR at 80 K. The λ max of light-adapted AR3 reconstituted in E. coli polar lipids at room temperature was found to be near 570 nm (data not shown) but the UV-visible absorption at 80 K has not yet been measured.…”

Isotope-edited IR spectroscopy for the study of membrane proteins

“…Its photocycle in particular has been studied in detail, in the context of the high-resolution structure of the protein [34]. Recent studies from the Braiman [35 ], Kandori [36 ] and Rothschild [37 ] groups (amongst others) utilizing isotope-edited FTIR demonstrate the advantageousness of the method. Rothschild and co-workers have studied the influence of incorporating selenomethionine in a cell-free expression system on the spectral properties of BR [37 ].…”