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A monomeric red fluorescent protein - PubMed

  • ️Tue Jan 01 2002

A monomeric red fluorescent protein

Robert E Campbell et al. Proc Natl Acad Sci U S A. 2002.

Abstract

All coelenterate fluorescent proteins cloned to date display some form of quaternary structure, including the weak tendency of Aequorea green fluorescent protein (GFP) to dimerize, the obligate dimerization of Renilla GFP, and the obligate tetramerization of the red fluorescent protein from Discosoma (DsRed). Although the weak dimerization of Aequorea GFP has not impeded its acceptance as an indispensable tool of cell biology, the obligate tetramerization of DsRed has greatly hindered its use as a genetically encoded fusion tag. We present here the stepwise evolution of DsRed to a dimer and then either to a genetic fusion of two copies of the protein, i.e., a tandem dimer, or to a true monomer designated mRFP1 (monomeric red fluorescent protein). Each subunit interface was disrupted by insertion of arginines, which initially crippled the resulting protein, but red fluorescence could be rescued by random and directed mutagenesis totaling 17 substitutions in the dimer and 33 in mRFP1. Fusions of the gap junction protein connexin43 to mRFP1 formed fully functional junctions, whereas analogous fusions to the tetramer and dimer failed. Although mRFP1 has somewhat lower extinction coefficient, quantum yield, and photostability than DsRed, mRFP1 matures >10 times faster, so that it shows similar brightness in living cells. In addition, the excitation and emission peaks of mRFP1, 584 and 607 nm, are approximately 25 nm red-shifted from DsRed, which should confer greater tissue penetration and spectral separation from autofluorescence and other fluorescent proteins.

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Figures

Figure 1
Figure 1

Graphical representation of the tetramer, dimer, and monomer of DsRed based on the x-ray crystal structure of DsRed (21). Residues 1–5 were not observed in the crystal structure (Protein Data Bank identification 1G7K) but have been arbitrarily appended for the sake of representation. The DsRed chromophore is represented in red, and the four chains of the dimer are labeled following the convention of Yarbrough et al. (21). (A) The tetramer of DsRed with all residues mutated in T1 indicated in green for external residues and blue for those internal to the β-barrel. (B) The AC dimer of DsRed with all mutations present in dimer2 represented as in A and the intersubunit linker present in tdimer2(12) shown as a dotted line. (C) The monomer of DsRed with all mutations present in mRFP1 represented as in A. This figure was produced with

molscript

(27).

Figure 2
Figure 2

Analytical ultracentrifugation analysis of DsRed, dimer2, and mRFP0.5a. The equilibrium radial absorbance profiles at 20,000 rpm were modeled with a theoretical curve that allowed only the molecular weight to vary. (A) The DsRed absorbance profile was best fit with an apparent molecular mass of 120 kDa, consistent with a tetramer. (B) The dimer2 absorbance profile was best fit with an apparent mass weight of 60 kDa, consistent with a dimer. (C) The mRFP0.5a absorbance profile was best fit with an apparent molecular mass of 32 kDa, consistent with a monomer containing an N-terminal polyhistidine affinity tag.

Figure 3
Figure 3

Fluorescence and absorption spectra of DsRed (A), T1 (B), dimer2 and tdimer2(12) (C), and mRFP1 (D). The absorbance spectrum is shown with a solid line, the excitation with a dotted line, and the emission with a dashed line.

Figure 4
Figure 4

Maturation of red fluorescence for DsRed, T1, dimer2, tdimer2(12), and mRFP1. Log-phase cultures of E. coli expressing the construct of interest were rapidly purified at 4°C, and beginning at 2 h postharvest their maturation at 37°C was monitored. The initial decrease in mRFP1 fluorescence is attributed to a slight quenching on warming from 4°C to 37°C.

Figure 5
Figure 5

HeLa cells expressing Cx43 fused with T1, dimer2, or mRFP1. (A, C, and E) Images were acquired with excitation at 568 nm (55 nm bandwidth) and emission at 653 nm (95 nm bandwidth) with additional transmitted light. Lucifer yellow fluorescence (B, D, and F) was acquired with excitation at 425 nm (45 nm bandpass) and emission at 535 nm (55 nm bandpass). (A) Two contacting cells transfected with Cx43-mRFP1 and connected by a single large gap junction. (B) One cell is microinjected with lucifer yellow at the point indicated by * and the dye quickly passes (1–2 s) to the adjacent cell. (C) Four neighboring cells transfected with Cx43-dimer2. The bright line between the two rightmost cells is the result of having two fluorescent membranes in contact and is not a gap junction. (D) As was observed about one-third of the time, microinjected dye is slowly passed to an adjacent cell. (E) Two adjacent cells transfected with Cx43-T1 and displaying the typical perinuclear localized aggregation. (F) No dye passed between neighboring cells.

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