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 but in addition 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-N15 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 H218O 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 chromophores 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.

Photochemical and Thermal Stability of Green and Blue Proteorhodopsins: Implications for Protein-Based Bioelectronic Devices

“…The cyclicity and thermal data presented in this work, although collected in a detergent environment, further demonstrate the differences between BPR and GPR 78,79. The reduced photochemical stability of BPR best exemplifies this difference and might be viewed to support those proposals that BPR is a regulatory or sensory protein 31,70.…”

Structural Changes in an Anion Channelrhodopsin: Formation of the K and L Intermediates at 80 K

“…These later bands might arise from the C=O stretch of the substituted Gln residue similar to assignment of Gln residues in proteorhodopsin in this region. 30 At 170 K, the magnitudes of several of the bands in this region in WT increase [(+)1748, (−)1742, (+)1724, and (−)1709 cm −1 ] and may therefore be associated with L formation at higher temperatures. The most prominent effect of the E68Q mutation at this temperature is the disappearance of the (+)1724 and (−)1709 cm −1 bands, which indicates that these bands may arise directly from the Glu68 residue.…”

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

“…1) showed that, like BR and all other archaeal rhodopsins studied, the initial photochemistry involved all-trans to 13-cis isomerization of the retinylidene chromophore (see for example recent FTIR studies on green and blue proteorhodopsin [30]). In agreement, resonance Raman (RRS) measurements using 785-nm near-infrared excitation revealed that the AR3 chromophore has an all-trans structure very similar to BR (manuscript in preparation, E.C.…”