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

Abstract

The photoisomerization of the retinal in bacteriorhodopsin is selective and efficient and yields perturbation of the protein structure within femtoseconds. The stored light energy in the primary intermediate is then used for the net translocation of a proton across the membrane in the microsecond to millisecond regime. This study is aimed at identifying how the protein changes on photoisomerization by using the O-H groups of threonines as internal probes. Polarized Fourier-transform IR spectroscopy of [3-(18)O]threonine-labeled and unlabeled bacteriorhodopsin indicates that 3 of the threonines (of a total of 18) change their hydrogen bonding. One is exchangeable in D(2)O, but two are not. A comprehensive mutation study indicates that the residues involved are Thr-89, Thr-17, and Thr-121 (or Thr-90). The perturbation of only three threonine side chains suggests that the structural alteration at this stage of the photocycle is local and specific. Furthermore, the structural change of Thr-17, which is located >11 A from the retinal chromophore, implicates a specific perturbation channel in the protein that accompanies the retinal motion.

Figure 1

Structure of BR (1BRX; ref. 10). (a) View from the cytoplasmic side along the membrane normal. (b) View from the C helix side along the membrane. The retinal chromophore is colored yellow in the space-filling model. Backbones of A–D helices (residues < 131) and E–G helices (residues ≥ 131) are colored blue and red, respectively. The side chains of the 11 threonines inside the membrane, at positions 17, 24, 46, 47, 55, 89, 90, 121, 142, 178, and 205, are represented by space-filling models. The other threonines are at positions 5, 67, 107, 128, 157, 170, and 247 in the loop and terminal regions.

Scheme 1

Structure of the retinal chromophore.

Figure 2

The K minus BR difference spectra of unlabeled (dotted lines) and [3-¹⁸O]threonine-labeled (solid lines) BR in the 3,700- to 2,000-cm⁻¹ region with the window-tilting angles (φ₀) of 0° (a) and 53.5° (b). The sample was hydrated with D₂O, and spectra were measured at 77 K. These frequencies cover the entire O-H and O-D stretches. One division of the y axis corresponds to 0.001 absorbance unit. Both dotted and solid lines overlap well except for three frequency region shown by arrows, indicating that three threonines change the stretching frequencies of their O-H (O-D) groups on photoisomerization. Diff. Abs., difference absorbance.

Figure 3

The K minus BR difference spectra of unlabeled (dotted lines) and [3-¹⁸O]threonine-labeled (solid lines) BR in the 3,530- to 3,350-cm⁻¹ region. The sample was hydrated with D₂O. The window-tilting angles (φ₀) are 0° (a), 17.8° (b), 35.7° (c), and 53.5° (d). One division of the y axis corresponds to 0.001 absorbance unit. Two peak pairs, 3,480 (+)/3,462 (−) cm⁻¹ and 3415 (+)/3402 (−) cm⁻¹, are able to be assigned for the O-H stretching vibrations of threonine side chains. Diff. Abs., difference absorbance.

Figure 4

The K minus BR difference spectra in the 3,530- to 3,350-cm⁻¹ region for the wild type (dotted lines) and the mutants (solid lines) T17V (a), T24V (b), T46V (c), T47V (d), T55V (e), T89S (f), T90V (g), T121V (h), T142N (i), T178N (j), and T205V (k). The sample was hydrated with D₂O, and the window-tilting angle (φ₀) is 53.5°. One division of the y axis corresponds to 0.001 absorbance unit.