Chiang, C.Y., Chen, Y.L., and Tsai, H.J. (2014) Different Visible Colors and Green Fluorescence Were Obtained from the Mutated Purple Chromoprotein Isolated from Sea Anemone. Marine biotechnology (New York, N.Y.). 16(4):436-46.
Green fluorescent protein (GFP)-like proteins have been studied with the aim of developing fluorescent proteins. Since the property of color variation is understudied, we isolated a novel GFP-like chromoprotein from the carpet anemone Stichodactyla haddoni, termed shCP. Its maximum absorption wavelength peak (λmax) is located at 574 nm, resulting in a purple color. The shCP protein consists of 227 amino acids (aa), sharing 96 % identity with the GFP-like chromoprotein of Heteractis crispa. We mutated aa residues to examine any alteration in color. When E63, the first aa of the chromophore, was replaced by serine (E63S), the λmax of the mutated protein shCP-E63S was shifted to 560 nm and exhibited a pink color. When Q39, T194, and I196, which reside in the surrounding 5 Å of the chromophore’s microenvironment, were mutated, we found that (1) the λmax of the mutated protein shCP-Q39S was shifted to 518 nm and exhibited a red color, (2) shCP-T194I exhibited a purple-blue color, and (3) an additional mutation at I196H of the mutated protein shCP-E63L exhibited green fluorescence. In contrast, when the aa located neither at the chromophore nor within its microenvironment were mutated, the resultant proteins shCP-L122H, -E138G, -S137D, -T95I, -D129N, -T194V, -E138Q, -G75E, -I183V, and -I70V never altered their purple color, suggesting that mutations at the shCP chromophore and the surrounding 5 Å microenvironment mostly control changes in color expression or cause fluorescence to develop. Additionally, we found that the cDNAs of shCP and its mutated varieties are faithfully and stably expressed both in Escherichia coli and zebrafish embryos.