Structural Change of Threonine 89 upon Photoisomerization in Bacteriorhodopsin As Revealed by Polarized FTIR Spectroscopy

Abstract

The all-trans to 13-cis photoisomerization of the retinal chromophore of bacteriorhodopsin occurs selectively, efficiently, and on an ultrafast time scale. The reaction is facilitated by the surrounding protein matrix which undergoes further structural changes during the proton-transporting reaction cycle. Low-temperature polarized Fourier transform infrared difference spectra between bacteriorhodopsin and the K intermediate provide the possibility to investigate such structural changes, by probing O-H and N-H stretching vibrations [Kandori, Kinoshita, Shichida, and Maeda (1998) J. Phys. Chem. B 102, 7899-7905]. The measurements of [3-18O]threonine-labeled bacteriorhodopsin revealed that one of the D2O-sensitive bands (2506 cm(-1) in bacteriorhodopsin and 2466 cm(-1) in the K intermediate, in D2O exhibited 18(O)-induced isotope shift. The O-H stretching vibrations of the threonine side chain correspond to 3378 cm(-1) in bacteriorhodopsin and to 3317 cm(-1) in the K intermediate, indicating that hydrogen bonding becomes stronger after the photoisomerization. The O-H stretch frequency of neat secondary alcohol is 3340-3355 cm(-1). The O-H stretch bands are preserved in the T46V, T90V, T142N, T178N, and T205V mutant proteins, but diminished in T89A and T89C, and slightly shifted in T89S. Thus, the observed O-H stretching vibration originates from Thr89. This is consistent with the atomic structure of this region, and the change of the S-H stretching vibration of the T89C mutant in the K intermediate [Kandori, Kinoshita, Shichida, Maeda, Needleman, and Lanyi (1998) J. Am. Chem. Soc. 120, 5828-5829]. We conclude that all-trans to 13-cis isomerization causes shortening of the hydrogen bond between the OH group of Thr89 and a carboxyl oxygen atom of Asp85.

Local and distant protein structural changes on photoisomerization of the retinal in bacteriorhodopsin

“…L- [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] O]threonine was synthesized as described (25). [3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18] O]threonine-labeled BR was prepared by growing Halobacterium salinarum (JW-3) in a defined medium similar to that described by Gochnauer and Kushner (26), except that the D-amino acids and NH 4 Cl were omitted and the L-threonine was replaced by 0.25 g͞liter L- [3-18 O]threonine.…”

“…Polarized FTIR spectroscopy was applied as described (23,25). A 120-l aliquot of the sample in 2 mM phosphate buffer (pH 7.0) was dried on a BaF 2 window with diameter of 18 mm.…”

Tight Asp-85–Thr-89 association during the pump switch of bacteriorhodopsin

“…Furthermore, combined studies of isotopelabeled and mutant BR can lead to identification of particular vibrational bands. In fact, by use of 3-18 O-threonine-labeled BR and mutation of individual threonine residues, we showed that among 18 threonines in BR, only three, Thr-89 (26), Thr-17, and Thr-121 (or Thr-90) (27), change their hydrogen bonds in the K intermediate.…”

“…) indicates a strong hydrogen bond (26). In K, a still lower stretching frequency (2,466 cm Ϫ1 ) corresponds to an even stronger hydrogen bond.…”

Early Photocycle Structural Changes in a Bacteriorhodopsin Mutant Engineered to Transmit Photosensory Signals

“…were assigned to the O-H stretching vibrations of Thr-17 and Thr-121, respectively (35,36). Corresponding bands seem to be present in BR-T. We tentatively assigned the band at 3518(Ϫ) cm Ϫ1 as the O-H stretch of introduced Thr-215 in BR-T.…”