Color transitions in coral's fluorescent proteins by site-directed mutagenesis

Abstract

Background: Green Fluorescent Protein (GFP) cloned from jellyfish Aequorea victoria and its homologs from corals Anthozoa have a great practical significance as in vivo markers of gene expression. Also, they are an interesting puzzle of protein science due to an unusual mechanism of chromophore formation and diversity of fluorescent colors. Fluorescent proteins can be subdivided into cyan (approximately 485 nm), green (approximately 505 nm), yellow (approximately 540 nm), and red (>580 nm) emitters.

Results: Here we applied site-directed mutagenesis in order to investigate the structural background of color variety and possibility of shifting between different types of fluorescence. First, a blue-shifted mutant of cyan amFP486 was generated. Second, it was established that cyan and green emitters can be modified so as to produce an intermediate spectrum of fluorescence. Third, the relationship between green and yellow fluorescence was inspected on closely homologous green zFP506 and yellow zFP538 proteins. The following transitions of colors were performed: yellow to green; yellow to dual color (green and yellow); and green to yellow. Fourth, we generated a mutant of cyan emitter dsFP483 that demonstrated dual color (cyan and red) fluorescence.

Conclusions: Several amino acid substitutions were found to strongly affect fluorescence maxima. Some positions primarily found by sequence comparison were proved to be crucial for fluorescence of particular color. These results are the first step towards predicting the color of natural GFP-like proteins corresponding to newly identified cDNAs from corals.

Figure 1

Multiple alignment of GFP and Anthozoa FPs. The numbering is according to GFP, the N- and C-termini of the proteins are not shown. Coral FPs are named using a nomenclature suggested in [7]. Each protein is designated by lowercase letters identifying the species (e.g., "z" for Zoanthus sp., "am" for Anemonia majano, "c" for Clavularia sp., "ds" for Discosoma sp., "dr" for Discosoma sp. red, "as" for Anemonia sulcatd), FP for fluorescent protein, and a number corresponding to the major emission maximum. Two proteins from Zoanthus and three from Discosoma are grouped and compared inside the groups: in the second/third protein of the group the amino acids identical to the corresponding ones in the first protein are represented by dashes. The protein names are shaded in colors corresponding to its fluorescence. The residues whose side chains form the interior of the β-can are shaded in blue (according to [6]). Introduced gaps are represented by dots. The residues that were mutated are shaded in pink. Red FP-specific Ser-68 and Ser/Thr-112 are shaded in red.

Figure 2

Excitation-emission spectra for mutants with altered fluorescent color. In some cases spectra for the wild type proteins are shown for comparison. Within each pair of lines the emission spectrum is the one at longer wavelengths. (A) Spectra for amFP486 wild type (dotted lines) and its blue shifted mutant Y66W (solid lines). (B) Spectra for mutants amFP486-A167M (solid lines) and zFP506-M167A (gray thick lines). (C) Spectra for green mutant zFP538-K65M (solid lines) and zFP506 wild type (gray thick lines). (D) Spectra for dual-color mutant zFP538-D68N. (E) Spectra for yellow mutant zFP506-A63G/N65K/N68D (solid lines) and zFP538 wild type (gray thick lines). (F) Spectral characteristics of the dual-color mutant protein dsFP483-N68S/I112S. Dotted line - double-peak fluorescence (excitation at 280 nm). Solid line - red fluorescence (excitation at 540 nm). Dashed line - excitation spectrum for red emission (at 620 nm).

Figure 3

Colonies of E. coli expressing wild type dsFP483 (wt) or its mutant N68S/I112S (mut) as viewed by fluorescence microscopy. (A) - using FITC filter set. (B) - using TRITC filter set. Colony expressing the wild type protein possesses only green fluorescence. In contrast, colony expressing the mutant protein is clearly visible in both filter set due to dual color (green and red) fluorescence of dsFP483-N68 S/I112S.