Diversity and evolution of coral fluorescent proteins

Abstract

GFP-like fluorescent proteins (FPs) are the key color determinants in reef-building corals (class Anthozoa, order Scleractinia) and are of considerable interest as potential genetically encoded fluorescent labels. Here we report 40 additional members of the GFP family from corals. There are three major paralogous lineages of coral FPs. One of them is retained in all sampled coral families and is responsible for the non-fluorescent purple-blue color, while each of the other two evolved a full complement of typical coral fluorescent colors (cyan, green, and red) and underwent sorting between coral groups. Among the newly cloned proteins are a "chromo-red" color type from Echinopora forskaliana (family Faviidae) and pink chromoprotein from Stylophora pistillata (Pocilloporidae), both evolving independently from the rest of coral chromoproteins. There are several cyan FPs that possess a novel kind of excitation spectrum indicating a neutral chromophore ground state, for which the residue E167 is responsible (numeration according to GFP from A. victoria). The chromoprotein from Acropora millepora is an unusual blue instead of purple, which is due to two mutations: S64C and S183T. We applied a novel probabilistic sampling approach to recreate the common ancestor of all coral FPs as well as the more derived common ancestor of three main fluorescent colors of the Faviina suborder. Both proteins were green such as found elsewhere outside class Anthozoa. Interestingly, a substantial fraction of the all-coral ancestral protein had a chromohore apparently locked in a non-fluorescent neutral state, which may reflect the transitional stage that enabled rapid color diversification early in the history of coral FPs. Our results highlight the extent of convergent or parallel evolution of the color diversity in corals, provide the foundation for experimental studies of evolutionary processes that led to color diversification, and enable a comparative analysis of structural determinants of different colors.

Figure 1. Excitation (solid lines) and emission (dashed lines) spectra of the newly cloned GFP-like proteins.

Horizontal axis: wavelength in nanometers, vertical axis: relative fluorescence amplitude.

Figure 2. Bayesian phylogenetic tree of the cnidarian fluorescent proteins; Arthropoda FPs are shown as an outgroup.

The edges with posterior probability less than 0.95 are collapsed. Black dots identify the proteins first described in this paper. Major clades and sub-clades are denoted, as well as the two reconstructed ancestral proteins (All-coral and All-Faviina). See legend for the color-coding of FP color classes. The position of meffGFP is tentative based on the short portion of its sequence that did not undergo gene conversion (see text for details). See Table S1 for the GenBank accession numbers corresponding to the protein names, and File S1 for the FASTA-formatted alignment of coding cDNA sequences.

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).

Figure 4. Positions of emission maxima in the mutated purple chromoprotein gfasCP in comparison to the blue amilCP.

Horizontal axis is wavelength in nanometers, the bars indicate the position of the absorption peak in the mutant. The colors of the bars approximately correspond to the colors of the mutants. Mutations S64C and S183T were found to be responsible for the blue color in amilCP (numeration according to GFP). Mutation I162L results in a very slowly maturing protein, hence pale color of the corresponding bar. This effect is rescued by any of the other two mutations.

Figure 5. Glutamic acid at position 167 (numeration according to GFP) determines the “neutral-chromophore” phenotype in novel cyan fluorescenr proteins.

A: Replacement of native E 167 by glycine in anobCFP leads to disappearance of 425 nm excitation peak and shift of both excitation and emission curves towards green. B: Reciprocal replacement G167E in a cyan protein meffCFP leads to the opposite results: virtually all the protein bulk started absorbing at 400 nm. Notably, the emission peak did not change.