Structural basis for the fast maturation of Arthropoda green fluorescent protein

Abstract

Since the cloning of Aequorea victoria green fluorescent protein (GFP) in 1992, a family of known GFP-like proteins has been growing rapidly. Today, it includes more than a hundred proteins with different spectral characteristics cloned from Cnidaria species. For some of these proteins, crystal structures have been solved, showing diversity in chromophore modifications and conformational states. However, we are still far from a complete understanding of the origin, functions and evolution of the GFP family. Novel proteins of the family were recently cloned from evolutionarily distant marine Copepoda species, phylum Arthropoda, demonstrating an extremely rapid generation of fluorescent signal. Here, we have generated a non-aggregating mutant of Copepoda fluorescent protein and solved its high-resolution crystal structure. It was found that the protein beta-barrel contains a pore, leading to the chromophore. Using site-directed mutagenesis, we showed that this feature is critical for the fast maturation of the chromophore.

Figure 1

Confocal microscopy of fluorescent protein expression in HeLa cells. (A) ppluGFP2 (green fluorescent protein from Pontellina plumata) forms needles 3 days after transfection. (B) ppluGFP2–β-actin fusion protein. (C) TurboGFP–β-actin fusion protein. (D) Enhanced green fluorescent protein–β-actin fusion protein.

Figure 2

Comparison of TurboGFP and enhanced green fluorescent protein maturation speed in developing Xenopus laevis embryos. At the stage of two blastomeres, embryos were microinjected with TurboGFP-C1 and pEGFP-C1 vectors. Living embryos were photographed from the animal pole side at the early and mid-gastrula stages. EGFP, enhanced green fluorescent protein.

Figure 3

TurboGFP crystal structure. (A) The overall structure of TurboGFP. Cα traces are in green, violet, red and blue. The chromophore is shown as green van der Waals spheres. (B–E) A pore leading to the TurboGFP chromophore. The chromophore is highlighted in green and Val 197 in red. (B) Protein surface (grey) is cut away to show the pore and the chromophore cavity. Sections of secondary structure elements are shown as yellow cartoons. Relevant water molecules are depicted as magenta spheres. (C–E) The pore remains unobstructed on tetramerization. (F,G) Contacts between TurboGFP monomers. Two TurboGFP monomers are shown as Cα traces, with contact residues shown as balls and sticks of the same colour as the particular monomer. For clarity, only one set of residues is labelled. (F) The more extensive contact area having mainly hydrophobic character is shown. (G) The less extensive contact area, composed of a mixture of polar and hydrophobic interactions is shown.

Figure 4

Comparison of refolding and maturation speed in vitro of EGFP, TurboGFP and TurboGFP-V197L. Normalized fluorescence recovery plots are shown. EGFP, violet lines; TurboGFP, green lines; TurboGFP-V197L, blue lines. See Methods and Table 1 for details. (A) Refolding kinetics. (B) Chromophore maturation kinetics.