underlying posterior distribution. Such a probabilistic mode of reconstruction has been proposed as a way to avoid bias towards higher stability and overall functional efficiency that could be expected in a consensus protein [34]. Using five samples, we expected to see the same phenotype in all the reconstructed variants, which would indicate that this phenotype represents a majority of all possible ancestral phenotypes with 95% confidence [35]. The sampled ancestral sequences corresponding to the all-coral ancestor differed between each other by 8–12%, all-Faviina sequences by 6–9% (Fig. 3 A, B). Despite these sequence differences, all the reconstructed variants exhibited practically identical fluorescence and absorbance phenotypes per ancestral node, with positions of the major peaks matching within 2 nm. This result indicates that the uncertainties of the ancestral sequence prediction did not affect the reconstructed ancestral phenotypes. All of the reconstructed ancestral proteins demonstrated green emission (Fig. 3 E ) with the maximum of 505–506 nm and mirror-image excitation spectrum peaking at 493–495 nm (Fig. 3 D). Interestingly however, the absorbance spectrum differed rather dramatically between the all-coral and all-Faviina ancestors (Fig. 3 C): whereas all-Faviina absorbance spectrum was very similar to the excitation spectrum, suggesting the presence of typical GFP- like chromophore in its anionic ground state [36,37], the absorbance spectrum of the all-coral ancestor featured a major peak at 375 nm that was practically not manifested in the excitation spectrum. This absorbance peak most likely corresponds to the chromophore in the neutral state, although it is more UV- shifted than in GFP from A. victoria (395 nm) or any of the cyan fluorescent proteins mentioned above (404 nm). Another distinc- Coral Fluorescent Proteins tive feature that may actually be related to the UV-shift is that in the all-coral ancestor this chromophore state is very low- fluorescent (hence the almost complete absence of the 375 nm peak in the excitation spectrum, Fig. 3D), perhaps due to the lack of the proton transfer pathway that enables fluorescence after absorption in the neutral state [37]. The low molar extinction coefficient at 493 nm (31,000–33,000 M21 for different variants) and low quantum yield (0.43–0.47) of the all-coral ancestor are ostensibly due to the large fraction of the protein being ‘‘locked’’ in the dark neutral state. The same parameters in the all-Faviina ancestor were on par with extant wild-type green proteins: its different sequence variants had molar extinction coefficient 88,000–100,000 M21 and quantum yields of 0.67–0.80. All the reconstructed protein were tetrameric or higher order oligomeric according to the semi-native electrophoresis [26]. Purple to blue shift in chromoproteins The chromoprotein amilCP is very similar to other coral chromoproteins in sequence; however, its absorption maximum (592 nm) is red-shifted by about 10 nm, making the protein appear blue instead of purple to the naked human eye. The closest homolog of amilCP is gfasCP, in comparison to which the amilCP protein has only four amino acid substitutions: S64C, I162L, S183T and S229P (numeration according to GFP from A. victoria). We investigated the effect of all combinations of these four mutations by introducing them into gfasCP and found that the blue phenotype was due to the substitutions at two sites: S64C and S183T (Fig. 4). The mutation at the fourth site (I162L) when introduced alone severely impaired the protein maturation: it took several days for soluble protein extract isolated after overnight Figure 3. Analysis of ancestral proteins. A: alignment of the amino acid sequences of the reconstructed ancestral variants, five corresponding to All-coral ancestor (all-cor, 0 to 4) and five corresponding to All-Faviina ancestor (all-fav, 0 to 4). B: Unrooted neighbor-joining tree illustrating the degree of divergence between the synthesized ancestral sequences. C–E: absorbance, excitation, and emission spectra of a representative all-coral ancestor (black curves) and all-Faviina ancestor (red curves). doi:10.1371/journal.pone.0002680.g003 PLoS ONE | www.plosone.org 7 July 2008 | Volume 3 | Issue 7 | e2680