title: Color-changing mutation in the E-F loop of proteorhodopsin.
abstract: It is usually assumed that only amino acids located near the retinal chromophore are responsible for color tuning of rhodopsins. However, we recently found that replacement of Ala178 with Arg in the E-F loop of proteorhodopsin (PR), an archaeal-type rhodopsin in marine bacteria, shifts the lambda(max) from 525 to 545 nm at neutral pH [Yoshitsugu, M., Shibata, M., Ikeda, D., Furutani, Y., and Kandori, H. (2008) Angew. Chem., Int. Ed. 47, 3923-3926]. Since the location of Ala178 is distant from the retinal chromophore (approximately 25 A), the molecular mechanism of the unusual mutation effect on color tuning is intriguing. Here we studied this mechanism by using additional mutations and some analytical methods. Introduction of Arg into the corresponding amino acid in bacteriorhodopsin (BR, M163R mutant) does not change the absorption spectra, indicating that the effect is specific to PR. Introduction of Arg into the A-B or C-D loop yields little (3 nm) or no color change, respectively. T177R and P180R mutants exhibited absorption spectra identical to that of the wild type, while N176R and S179R mutants exhibit lambda(max) values of 528 and 535 nm, respectively. Therefore, the observed color change is position-specific, being fully effective at position 178 and half-effective at position 179. Salt affects the absorption spectra of wild-type and A178R PR similarly. FTIR spectroscopy at 77 K indicated similar chromophore structures for wild-type and A178R PR, and A178R PR pumps protons normally. We infer that the E-F loop has a unique structure in PR and the mutation of Ala178 disrupts the structure that includes the transmembrane region, leading to the observed changes in color and pK(a).
authors.affiliation: Department of Frontier Materials, Nagoya Institute of Technology, Showa-ku, Nagoya 466-8555, Japan.
authors.forename: Maiko
authors.initials: M
authors.lastname: Yoshitsugu
authors.forename: Junya
authors.initials: J
authors.lastname: Yamada
authors.forename: Hideki
authors.initials: H
authors.lastname: Kandori