V108M mutant of pharaonis phoborhodopsin: substitution caused no absorption change but affected its M-state.

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Abstract
Crystallographic data reveal that Met-118 in bacteriorhodopsin (bR) contacts directly with the C9 methyl group of retinal, and Khorana et al. [J. Biol. Chem. 268, 20305-20311 (1993)] suggest that this contact may regulate the absorption maximum (λmax). We have replaced the amino acid (Val-108) corresponding to Met-118 of bR by methionine in pharaonis phoborhodopsin (ppR), whose λmax is ca. 500 nm, while those of other bacterial rhodopsins such as bR, halorhodopsin, and sensory rhodopsin are red-shifted by 60–90 nm. By flashphotolysis measurement, we could not recognize a large spectral red-shift of the V108M mutant. On the other hand, the decay of ppRM (M-intermediate) of the mutant was approximately three times as fast as that of wild-type, and an M-like intermediate (M') whose λmax is blue-shifted by 60 nm from that of M became appreciable. The replacement abolished the shoulder of the ppRM spectrum. From these findings, we infer that the distance between the retinal and the 108-position in ppR is relatively long, and that in the M-state this distance is shortened.

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