Different structural changes occur in blue- and green-proteorhodopsins during the primary photoreaction

Abstract

We examine the structural changes during the primary photoreaction in blue-absorbing proteorhodopsin (BPR), a light-driven retinylidene proton pump, using low-temperature FTIR difference spectroscopy. Comparison of the light-induced BPR difference spectrum recorded at 80 K to that of green-absorbing proteorhodopsin (GPR) reveals that there are several differences in the BPR and GPR primary photoreactions despite the similar structure of the retinal chromophore and all-trans --> 13-cis isomerization. Strong bands near 1700 cm(-1) assigned previously to a change in hydrogen bonding of Asn230 in GPR are still present in BPR. However, additional bands in the same region are assigned on the basis of site-directed mutagenesis to changes occurring in Gln105. In the amide II region, bands are assigned on the basis of total (15)N labeling to structural changes of the protein backbone, although no such bands were previously observed for GPR. A band at 3642 cm(-1) in BPR, assigned to the OH stretching mode of a water molecule on the basis of H2(18)O substitution, appears at a different frequency than a band at 3626 cm(-1) previously assigned to a water molecule in GPR. However, the substitution of Gln105 for Leu105 in BPR leads to the appearance of both bands at 3642 and 3626 cm(-1), indicating the waters assigned in BPR and GPR exist in separate distinct locations and can coexist in the GPR-like Q105L mutant of BPR. These results indicate that there exist significant differences in the conformational changes occurring in these two types proteorhodopsin during the initial photoreaction despite their similar chromophore structures, which might reflect a different arrangement of water in the active site as well as substitution of a hydrophilic for hydrophobic residue at residue 105.

Figure 1

Comparison of FTIR difference spectra recorded at 80 K for BPR (top) and GPR (bottom) in the 900 – 1800 cm⁻¹ spectral region. The spectra were recorded at 2 cm⁻¹ spectral resolution. Each spectrum represents the average of at least 24 difference spectra, each consisting of 1000 individual scans. Spacing of Y-axis (difference absorbance) markers correspond to 0.5 × 10⁻⁴ OD. Only the frequency of bands discussed in paper in this and subsequent figures are labeled.

Figure 2

FTIR difference spectra recorded at 80K and 2 cm⁻¹ resolution for wild-type BPR containing unlabeled chromophore in H₂O, C15D labeled chromophore in H₂O, unlabeled chromophore with total-¹⁵N labeled protein, unlabeled chromophore in D₂O, and C15D labeled chromophore in D₂O. Spacing of Y-axis (difference absorbance) markers correspond to 0.2 × 10⁻⁴ OD.

Figure 3

FTIR difference spectra recorded at 80K and 2 cm⁻¹ resolution for wild-type BPR containing unlabeled chromophore in H₂O, C15D labeled chromophore in H₂O, unlabeled chromophore with total-¹⁵N labeled protein, unlabeled chromophore in D₂O, and C15D labeled chromophore in D₂O. Spacing of Y-axis (difference absorbance) markers correspond to 0.2 × 10⁻⁴ OD.

Figure 4

Comparison of the WT and N230S BPR difference spectra recorded at 80 K in the 1750 - 1560 cm⁻¹ region in H₂O (top) and comparison of the BPR N230S difference spectra in H₂O and D₂O (bottom). Spacing of Y-axis (difference absorbance) markers correspond to 0.2 × 10⁻⁴ OD.

Figure 5

Comparison of the WT GPR and Q105L BPR 80K FTIR difference spectra in the 1800 - 900 cm⁻¹ region. (Inset) Comparison of the wild-type BPR and Q105L BPR 80K difference spectra in the 1750 - 1560 cm⁻¹ region. Spacing of Y-axis (difference absorbance) markers correspond to 0.2 × 10⁻⁴ OD.

Figure 6

Comparison of the WT BPR, WT GPR, and Q105L 80K FTIR difference spectra in the 3700 - 3550 cm⁻¹ region: (top) WT BPR in H₂O (solid line) and H₂¹⁸O (dotted line); (middle) WT GPR in H₂O (solid line) and H₂¹⁸O (dotted line); (bottom) Q105L BPR in H₂O (solid line) and H₂¹⁸O (dotted line). Spacing of Y-axis (difference absorbance) markers correspond to 0.1 × 10⁻⁴ OD.

Figure 7

Hypothesized positions of residues Gln105, Asn230, and Tyr204 relative to the retinal chromophore and protonated Schiff base. The figure was created using PyMol software (DeLano, W. L. (2003) DeLano Scientific LLC, South San Francisco, CA 94080) from the PDB file 1JGJ by substituting the specific BPR residues shown onto the SRII crystal structure based on recent findings (54) as discussed in text. The BPR residues were imported to minimize steric interactions with the surrounding residues only. This model is only presented to provide a possible pathway and does not represent the free-energy minima of the protein.