A Color-Determining Amino Acid Residue of Proteorhodopsin

Proteorhodopsin (PR) is a light-driven proton pump found in marine bacteria. More than 1000 PRs are classified as blue-absorbing (λ_max ∼ 490 nm) and green-absorbing (λ_max ∼ 525 nm) PRs. The color determinant is known to be at position 105, where blue-absorbing and green-absorbing PRs possess Gln and Leu, respectively. This suggests hydrophobicity at position 105 plays a key role in color tuning. Here we successfully introduced 19 amino acid residues into position 105 of green-absorbing PR in the membrane environment and investigated the absorption properties. High-performance liquid chromatography analysis shows that the isomeric composition of the all-<i>trans</i> form is >70% for all mutants, indicating little influence of different isomers on color tuning. Absorption spectra of the wild-type and 19 mutant proteins were well-characterized by the pH-dependent equilibria of the protonated and deprotonated counterion (Asp97) of the Schiff base, whereas the λ_max values of these two states and the p<i>K</i>_a value differed significantly among mutants. Although Gln and Leu are hydrophilic and hydrophobic residues, respectively, the λ_max values of the two states and the p<i>K</i>_a value did not correlate with the hydropathy index of residues. In contrast, the λ_max and p<i>K</i>_a were correlated with the volume of residues, though Gln and Leu possess similar volumes. This observation concludes that the λ_max and p<i>K</i>_a of Asp97 are determined by local and specific interactions in the Schiff base moiety, in which the volume of the residue at position 105 is more influential than its hydrophobicity. We suggest that the hydrogen-bonding network in the Schiff base moiety plays a key role in the λ_max and p<i>K</i>_a of Asp97, and the hydrogen-bonding network is significantly perturbed by large amino acid residues but may be preserved by additional water molecule(s) for small amino acid residues at position 105