Mechanism of the Irreversible Transition from Pentamer to Monomer at pH 2 in a Blue Proteorhodopsin.

Proteorhodopsin (PR) is a light-driven proton pump found in marine bacteria, and thousands of PRs are classified as blue-absorbing PRs (BPR; λmax ∼ 490 nm) and green-absorbing PRs (GPR; λmax ∼ 525 nm). We previously converted BPR into GPR using an anomalous pH effect, which was achieved by an irreversible process at around pH 2. Recent size-exclusion chromatography (SEC) and atomic force microscopy (AFM) analyses of BPR from Vibrio califitulae (VcBPR) revealed the anomalous pH effect owing to the irreversible transition from pentamer to monomer. Different pKa values of the Schiff base counterion between pentamer and monomer lead to different colors at the same pH. Here, we incorporate systematic mutation into VcBPR and examine the anomalous pH effect. The anomalous pH effect was observed for the mutants of key residues near the retinal chromophore such as D76N, D206N, and Q84L, indicating that the Schiff base counterions and the L/Q switch do not affect the irreversible transition from pentamer to monomer at pH ∼ 2. We then focus on the two specific interactions at the intermonomer interface in a pentamer, E29/R30/D31 and W13/H54. Single mutants such as E29Q, R30A, W13A, and H54A and the wild type (WT) exhibited an anomalous pH effect. In contrast, the anomalous pH effect was lost for E29Q/H54A, R30A/H54A, and W13A/E29Q. Size-exclusion chromatography (SEC) and atomic force microscopy (AFM) measurements showed monomer forms in the original states of the double mutants, being a clear contrast to the pentamer forms of all single mutants in the original states. It was concluded that the pentamer structure of VcBPR was stabilized by an electrostatic interaction in the E29/R30/D31 region and a hydrogen-bonding interaction in the W13/H54 region, which was disrupted at pH 2 and converted into monomers.

Molecular Origin of the Anomalous pH Effect in Blue Proteorhodopsin.

Proteorhodopsin (PR) is a light-driven proton pump found in marine bacteria, and thousands of PRs are classified into blue-absorbing PR (BPR; λmax ∼ 490 nm) and green-absorbing PR (GPR; λmax ∼ 525 nm). We previously presented conversion of BPR into GPR using the anomalous pH effect. When we lowered the pH of a BPR to pH 2 and returned to pH 7, the protein absorbs green light. This suggests the existence of the critical point of the irreversible process at around pH 2, but the mechanism of anomalous pH effect was fully unknown. The present size exclusion chromatography (SEC) and atomic force microscope (AFM) analysis of BPR from Vibrio califitulae (VcBPR) revealed the anomalous pH effect because of the conversion from pentamer to monomer. The different pKa of the Schiff base counterion between pentamer and monomer leads to different colors at the same pH.