Retracing evolution of red fluorescence in GFP-like proteins from Faviina corals

Abstract

Proteins of the green fluorescent protein family represent a convenient experimental model to study evolution of novelty at the molecular level. Here, we focus on the origin of Kaede-like red fluorescent proteins characteristic of the corals of the Faviina suborder. We demonstrate, using an original approach involving resurrection and analysis of the library of possible evolutionary intermediates, that it takes on the order of 12 mutations, some of which strongly interact epistatically, to fully recapitulate the evolution of a red fluorescent phenotype from the ancestral green. Five of the identified mutations would not have been found without the help of ancestral reconstruction, because the corresponding site states are shared between extant red and green proteins due to their recent descent from a dual-function common ancestor. Seven of the 12 mutations affect residues that are not in close contact with the chromophore and thus must exert their effect indirectly through adjustments of the overall protein fold; the relevance of these mutations could not have been anticipated from the purely theoretical analysis of the protein's structure. Our results introduce a powerful experimental approach for comparative analysis of functional specificity in protein families even in the cases of pronounced epistasis, provide foundation for the detailed studies of evolutionary trajectories leading to novelty and complexity, and will help rational modification of existing fluorescent labels.

FIG. 1.

The need for vertical comparative analysis. (A) A typical phylogenetic relationship suggesting that a new function (red) evolved from the ancestral (green). E1, E2, and E3 designate extant proteins. (B) A case when the new function evolved after gene duplication. The black hashes represent neutral mutations and the colored hashes mutations required for the new function. Comparison of E2 with the extant protein E1 captures both essential mutations, but comparison to their common ancestor (A1) is more efficient because it includes less neutral mutations. (C) A scenario when the new function evolved prior to gene duplication. In this case, comparing E1 and E2 does not capture all the essential mutations, unless g1 is a reversal of r1. Instead, E2 should be compared with the deeper ancestral node A2. (D) Phylogenetic tree of GFP-like proteins from Faviina corals drawn on a Petri dish using bacteria-expressing extant and ancestral proteins (Ugalde et al. 2004). The common ancestor of greens and reds (r/g) is a dual-function orange. The evolution of the red color, therefore, should be traced between the extant red and the common green ancestor of all coral colors (a).

FIG. 2.

Simulated analysis of transitional libraries under different epistasis scenarios. Horizontal axis: number of clones characterized from the transitional library, vertical axis: causal mutation discovery rate, averaged over 100 replicates. The epistasis scenarios were as follows. “6”: a single group of six epistatically interacting mutations, that is, all six are required for the phenotype change. “3,3”: two groups of three epistatically interacting mutations with no epistasis between the groups, that is, the phenotype changes if all members of any one group are present. “3”: one group of three interacting mutations. “1,1,1”: three mutations with purely additive effects (no epistasis). We also performed a “sequencing budget” flavor of the analysis, where we used only 67 of all clones exhibiting ancestral phenotype in Fisher's test, as in our real study. These additional curves are designated “6 (67)” and “3,3 (67)”. The “sequencing budget” curves for other scenarios were identical to the full-analysis ones.

FIG. 3.

Changes in fluorescence of the ancestral protein as candidate color-changing mutations are introduced into it in the order of decreasing association with red color in the transitional library analysis. The graphs are normalized fluorescence spectra of expressing bacterial colonies after 120-min UV-B exposure; the horizontal axis is wavelength in nanometers. Green fluorescence corresponds to the peak at 500–520 nm and red to the peak at 575–580 nm. The numbering of mutated sites is according to the Aequorea victoria GFP sequence (Prasher et al. 1992) within a familywide alignment (Alieva et al. 2008); DelY stands for the deletion of a tyrosine between positions 227 and 228. (A,B) Introduction of mutations for which the association was significant at 0.05 level results in an 11-mutation clone that is almost as red as the extant red protein R1-2. (C) Adding different combinations of mutations that are significant at the 0.1 level meets and surpasses the red maturation efficiency of R1–2. (D) Visual appearance of bacteria-expressing mutant proteins that retrace the green-to-red evolutionary transition, under UV-A light.

FIG. 4.

Effect of individual mutation reversals in the ancestral clone bearing 11 candidate mutations (“11-mutation ancestor”). The graphs are normalized fluorescence spectra; the horizontal axis is wavelength in nanometers. (A) Large-effect mutations. (B) Mutations with lesser effect (“fine tuning”). Note that reversal of mutation at site 87 does not affect fluorescence, and hence, this mutation is not required for the red color.

FIG. 5.

Effect of mutations for which the association study strongly suggested preference toward the ancestral state in the redder clones. The graphs are normalized fluorescence spectra; the horizontal axis is wavelength in nanometers; dashed curves correspond to proteins with the mutation in question in the ancestral state. (A,B) mutation D11V is always deleterious for the red color, suppressing red fluorescence (575–580 nm) and promoting green (515–520 nm). (C,D) Mutation A63V exhibits sign epistasis (Weinreich et al. 2005): It is deleterious at less advanced evolutionary stage (C) but is beneficial later (D).

FIG. 6.

Distribution of the sites responsible for the red fluorescence, as suggested by this study, in a Kaede-type red fluorescent protein EosFP (Nienhaus et al. 2005). (A) Side chains that directly contribute to the chromophore or its environment are shown in yellow. Two residues in gray, with outward-directed side chains, most likely exert their effects through adjusting the positions of the yellow ones. The invariant chromophore portion (not including the part contributed by His65) is shown in red. (B) Side chains of the residues with indirect effect (gray). The chromophore is shown in red. Yellow side chains belong to the two evolutionarily conserved residues (Glu-222 and His-203); it is likely that mutations at the gray sites result in adjustment of positions of these side chains relative to the chromophore.