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Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral - PubMed

  • ️Sat Jan 01 2000

Biochemistry, mutagenesis, and oligomerization of DsRed, a red fluorescent protein from coral

G S Baird et al. Proc Natl Acad Sci U S A. 2000.

Abstract

DsRed is a recently cloned 28-kDa fluorescent protein responsible for the red coloration around the oral disk of a coral of the Discosoma genus. DsRed has attracted tremendous interest as a potential expression tracer and fusion partner that would be complementary to the homologous green fluorescent protein from Aequorea, but very little is known of the biochemistry of DsRed. We now show that DsRed has a much higher extinction coefficient and quantum yield than previously reported, plus excellent resistance to pH extremes and photobleaching. In addition, its 583-nm emission maximum can be further shifted to 602 nm by mutation of Lys-83 to Met. However, DsRed has major drawbacks, such as strong oligomerization and slow maturation. Analytical ultracentrifugation proves DsRed to be an obligate tetramer in vitro, and fluorescence resonance energy transfer measurements and yeast two-hybrid assays verify oligomerization in live cells. Also, DsRed takes days to ripen fully from green to red in vitro or in vivo, and mutations such as Lys-83 to Arg prevent the color change. Many potential cell biological applications of DsRed will require suppression of the tetramerization and acceleration of the maturation.

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Figures

Figure 1
Figure 1

DsRed maturation proceeds through a green intermediate. (A) Excitation spectrum (dashed line, emission collected at 500 nm) and emission spectrum (solid line, excitation at 475 nm) of bacterially expressed DsRed taken immediately after purification. (B) Time course of green to red conversion. Open symbols indicate green fluorescence (475-nm excitation, 500-nm emission). Filled symbols indicate red fluorescence (558-nm excitation, 584-nm emission). Squares and circles denote two separate trials. Each curve has been normalized by its maximum value to permit comparison of the time courses. The maximum green fluorescence is <1% of the maximum red fluorescence.

Figure 2
Figure 2

Excitation and emission spectra of DsRed (solid lines), as well as emission spectra of rhodamine B (dotted line) and rhodamine 101 (dashed line). All emission spectra were taken from solutions with equal absorbances at the excitation wavelength, 551.5 nm. Emission was monitored at 583 nm for the DsRed excitation spectrum. DsRed was in an aqueous solution buffered with 10 mM Tris, pH 8.5; rhodamines were in ethanol.

Figure 3
Figure 3

pH dependence of DsRed fluorescence and absorbance. (A) 583-nm emission monitored with 558-nm excitation. (B) 558-nm excitation monitored with emission at 583 nm. (C) Absorbance at 552 nm. (D) Excitation spectra of DsRed (emission monitored at 583 nm) at pH 8.8, 4.5, and 4 showing spectral shape change.

Figure 4
Figure 4

Excitation and emission spectra of DsRed (heavy solid line) and mutants K83R (thin solid line) and K83 M (dotted line). For each excitation spectrum, emission was monitored at the emission maximum, and vice versa.

Figure 5
Figure 5

Oligomerization of unboiled, polyhistidine-tagged fluorescent proteins monitored by electrophoresis in 15% polyacrylamide gels. Lane A: Broad range prestained protein standard (Bio-Rad). Lane B: DsRed. Lane C: K83R mutant of DsRed. Lane D: Wild-type Aequorea GFP.

Figure 6
Figure 6

Oligomerization of DsRed as shown by analytical ultracentrifugation. (A) Equilibrium radial absorbance profile (circles) measured at 20,000 rpm, overlaid by theoretical curve fits for species ranging from monomer to pentamer. The curves were globally fitted to data at 10,000, 14,000, and 20,000 rpm, although only the latter data are presented here. (B) Residual errors from the theoretical fits, illustrating that a tetramer (gray solid line) fits the experimental data better than monomer, dimer, trimer, or pentamer. (C) Residual errors on an expanded scale comparing simple tetramer (gray solid line, same values as in B) with a model allowing the tetramer to dimerize to an octamer (black solid line) with a Kd of 39 μM, i.e., [octamer] = [tetramer]2/(39 μM). The variance for the tetramer + octamer model was 1.18 × 10−5, somewhat better than that for the tetramer alone, 1.95 × 10−5.

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References

    1. Tsien R Y. Annu Rev Biochem. 1998;67:509–544. - PubMed
    1. Matz M V, Fradkov A F, Labas Y A, Savitsky A P, Zaraisky A G, Markelov M L, Lukyanov S A. Nat Biotechnol. 1999;17:969–973. - PubMed
    1. Gross L A, Baird G S, Hoffman R C, Baldridge K K, Tsien R Y. Proc Natl Acad Sci USA. 2000;97:11990–11995. - PMC - PubMed
    1. Heim R, Tsien R Y. Curr Biol. 1996;6:178–182. - PubMed
    1. Baird G S, Zacharias D A, Tsien R Y. Proc Natl Acad Sci USA. 1999;96:11241–11246. - PMC - PubMed

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