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 2O, 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 Å from the retinal chromophore, implicates a specific perturbation channel in the protein that accompanies the retinal motion. B acteriorhodopsin (BR) is a light-driven proton pump inHalobacterium salinarum that contains all-trans retinal as chromophore (1-4). Its tertiary structure has been determined recently by cryoelectron microscopy (5-7) and x-ray crystallography (8-13). The retinal binds covalently to Lys-216 through a protonated Schiff base linkage. Absorption of light triggers a cyclic reaction that comprises a series of intermediates, designated as the J, K, KL, L, M, N, and O states (1-4). Protein structural changes in these intermediate states cause proton translocation across the protein, and their mechanism is the central question in current studies of BR (14).The all-trans to 13-cis photoisomerization leads to the formation of the primary K intermediate (15)(16)(17). It is well known that photoisomerization in BR is highly selective and efficient. In solution, the photoproduct of all-trans retinal with protonated Schiff base is mainly 11-cis [82% (vol/vol) 11-cis͞6% (vol/vol) 13-cis͞12% (vol/vol) 9-cis in methanol; ref. 18], whereas in BR, the photoproduct is 100% (vol͞vol) 13-cis. The quantum efficiency for isomerization in BR (Ϸ0.6; refs. 19 and 20) is much higher than for retinal in solution (0.13 in methanol; ref. 18). This efficiency is correlated closely with the rate constant of the isomerization, because it occurs on the femtosecond time scale. The protein environment thus certainly facilitates the specificity of the reaction of the retinal chromophore.How does the highly selective and efficient isomerization occur in BR? How does the protein respond to the chromophore motion? To address these questions, structural analysis of the primary intermediates is essential. Cryoelectron microscopy of the K intermediate reported that the structural change at this stage is not resolved with 3.5-Å resolution (21). More recent analysis of the K intermediate by x-ray crystallography identified few...

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

“…2 originates from Thr-89. Next, we tested this tentative assignment by use of mutants as we did previously (26,27). In the present study, we tested four mutants: two are at position 89 (T89A and T89S) and two are at other locations (T46V and T205V).…”

“…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.…”

Spectral Signatures of the Pentagonal Water Cluster in Bacteriorhodopsin

“…[16] Because W402 is hydrogen-bonded to the positively charged Schiff base and to the two negative carboxylates Asp85 and Asp212 it is the most likely origin of this broad absorbance. [17,6,18,8,19,9,16] Computer simulations can relate such spectra to structural and functional models via combined quantum mechanical/molecular mechanical (QM/MM) calculations, [20,21,[10][11][12] which treat the region of interest with quantum mechanical electronic structure methods, whereas the surrounding environment is described by a molecular mechanical force field. In pioneering static QM/MM studies Hayashi et al determined harmonic frequencies for deuterated water molecules W401 and W402 and the NÀD stretch of the Schiff base, [20,21] which were found to Figure 1.…”

Conformational Coupling between the Cytoplasmic Carboxylic Acid and the Retinal in a Fungal Light-Driven Proton Pump

“…In the past, we have characterized the hydrogen-bonded networks in the protein moiety of various rhodopsins through the analysis of the X-D stretching vibrations, which appear in the 2800-1800 cm -1 region (13,16,(35)(36)(37)(38). Because of strong infrared absorption of water, it is hard to observe the O-H and N-H stretching vibrations under strongly hydrogen bonded conditions when the sample is hydrated with H 2 O.…”