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  • Title: Importance of alanine at position 178 in proteorhodopsin for absorption of prevalent ambient light in the marine environment.
    Author: Yamada K, Kawanabe A, Kandori H.
    Journal: Biochemistry; 2010 Mar 23; 49(11):2416-23. PubMed ID: 20170125.
    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. A recent mutation study revealed that the observed color change was highly specific to position 178 [Yoshitsugu, M., Yamada, J., and Kandori, H. (2009) Biochemistry 48, 4324-4330]. Thus, in the study presented here, we replaced Ala178 with 19 different amino acids and measured the absorption spectra and the pK(a) of the Schiff base counterion, Asp97. Most of the mutants exhibited a spectral red shift and increased pK(a) of Asp97. None of charged amino acids at position 178 influences color tuning of PR specifically, being similar to the Arg replacement studied earlier. Only Cys and Thr replacements exhibited color and a pK(a) similar to that of the wild type. Ser, Val, and Gly mutants behave like the wild type only with respect to the lambda(max) of the species with deprotonated Asp97. We conclude that the E-F loop region contains a unique structure in PR, disruption of which causes large-scale rearrangement of alpha-helices. Ala178 in PR contributes to the blue-shifted absorption (525 nm at neutral pH) and lowering of the counterion pK(a), which is important for proton-pump function in the marine environment, even though its position is far removed from the chromophore binding domain.
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