Figure 1.
Source organism and phylogenetic relationships of the new proteins.
A: Pontella mimocerami illuminated and imaged under white light. Note the blue non-fluorescent coloration, which is typical of many pontellid species. B: Same specimens under blue light showing the bright green fluorescence. On panels A and B, female is on the left, male is on the right. C: Bayesian phylogeny of the protein-coding sequences of all known GFPs from copepods of the Pontellidae family, rooted with cpGFP from Chiridius poppei, a copepod from the family Aetideidae. Scale bar: 0.05 substitutions per nucleotide site. The posterior probability at each node is 1.00 except where indicated. The two new proteins are indicated by green rectangles.
Figure 2.
Spectroscopic properties of the new proteins.
A: Normalized excitation (dashed line) and emission (solid line) curves of purified pmimGFP1 and pmimGFP2 (the curves for these two proteins are identical). Horizontal axis: wavelength in nanometers; vertical axis: fluorescence amplitude. B: Comparison of extinction, at each protein's own absorption maximum, between EGFP and the new proteins. C: Comparison of quantum yields of fluorescence (QY) between EGFP, new proteins, and the non-aggregating mutant of pmimGFP1.
Figure 3.
Aggregation and oligomerization analysis of the recombinant proteins.
A. Recombinant fluorescent proteins electrophoresed on a SDS-containing gel without prior heating and viewed with UV illumination. pmimGFP1 fluoresces brightly, pmimGFP2 is also visible, but is very faint. rGFP is a monomer, DsRed is a tetramer (indicated by an arrow). B. The same gel as in figure 3A, but imaged with Coomassie stain. rGFP, pmimGFP1, and pmimGFP 2 are all susceptible to partial denaturation under the running conditions and therefore show non-fluorescent bands at ∼25 kD, the expected size of a fully denatured protein. DsRed, the tetrameric standard, retains its multimeric state. C. The same samples as in figures A and B, but electrophoresed in fully denaturing conditions (before loading, the samples were boiled 5 min) followed by Coomassie stain. All four samples show the single major band corresponding to the denatured protein at about 25 kD. D. Removal of N-terminal positive charges reduces aggregation. Lane 1 is the wild type pmimGFP1, lane 2 K5E mutant, lane 3 is K5T mutant, and lane 4 is a triple mutant K5T, C8S, and R9A. Arrow indicates tetramer mobility.
Figure 4.
pH (A) and photostability (B) of novel copepod proteins and of the non-aggregating mutant of pmimGFP1.
On both panels, analogous measurements of commercially available recombinant EGFP protein are presented as a reference. On panel B, the horizontal axis is time of illumination under the fluorescence microscope, and the scale of the vertical axis is logarithmic. On both panels, each point represents an average of three replicate measurements.