Chemical nature of the light emitter of the Aequorea green fluorescent protein - PubMed (original) (raw)

Chemical nature of the light emitter of the Aequorea green fluorescent protein

H Niwa et al. Proc Natl Acad Sci U S A. 1996.

Abstract

The jellyfish Aequorea victoria possesses in the margin of its umbrella a green fluorescent protein (GFP, 27 kDa) that serves as the ultimate light emitter in the bioluminescence reaction of the animal. The protein is made up of 238 amino acid residues in a single polypeptide chain and produces a greenish fluorescence (lambda max = 508 nm) when irradiated with long ultraviolet light. The fluorescence is due to the presence of a chromophore consisting of an imidazolone ring, formed by a post-translational modification of the tripeptide -Ser65-Tyr66-Gly67-. GFP has been used extensively as a reporter protein for monitoring gene expression in eukaryotic and prokaryotic cells, but relatively little is known about the chemical mechanism by which fluorescence is produced. To obtain a better understanding of this problem, we studied a peptide fragment of GFP bearing the chromophore and a synthetic model compound of the chromophore. The results indicate that the GFP chromophore consists of an imidazolone ring structure and that the light emitter is the singlet excited state of the phenolate anion of the chromophore. Further, the light emission is highly dependent on the microenvironment around the chromophore and that inhibition of isomerization of the exo-methylene double bond of the chromophore accounts for its efficient light emission.

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Figures

Scheme I

Scheme I

Figure 1

Figure 1

Scheme for the formation of the chromophore in_Aequorea_ GFP. (A) Partial amino acid sequence of the N-terminal region of GFP showing the amino acid residues involved (underlined) in chromophore formation. (B) Dehydration–dehydrogenation mechanism for the formation of the chromophore.

Figure 2

Figure 2

Linear mode MALDI–TOF mass spectrum (Inset), MALDI–PSD fragment ion mass spectrum of the isolated lysyl endopeptidase fragment of GFP and proposed fragmentation products of the chromophore with their assigned masses. Details of the mass spectroscopic procedures are described in the text.

Figure 3

Figure 3

UV–visible absorption spectra of compound1 in DMSO. Traces: a, compound 1 in DMSO (neutral); b, compound 1 in DMSO containing 1 M HCl aqueous, 5% (vol/vol) (acidic); c, compound 1 in DMSO containing 1 M NaOH aqueous, 5% (vol/vol) (basic). Concentration of compound1 = 5.0 × 10−5 M.

Figure 4

Figure 4

UV–visible absorption spectra of lysyl endopeptidase digest of GFP (A) and compound 1 (B). Traces: a, GFP digest in 0.1 M NaOH; b, GFP digest in 0.1 M HCl; c, compound 1 in 2-propanol containing 0.1 M NaOH aqueous solution, 2.5%, (vol/vol); d, compound 1 in 2-propanol containing 0.1 M HCl aqueous solution, 2.5% (vol/vol). Estimated original concentration of GFP = 4.8 × 10−6 M; concentration of compound 1 = 5.0 × 10−5 M.

Figure 5

Figure 5

Fluorescence emission spectra of lysyl endopeptidase digest of GFP and compound 1 in ethanol glass at 77 K. Traces: a, digest of GFP in ethanol containing 0.1 M NaOH aqueous solution, 1% (vol/vol); b, compound 1 dissolved in ethanol containing 0.1 M NaOH aqueous solution, [% (vol/vol)] Estimated original concentration of GFP = 5.5 × 10−7 M; concentration of compound 1 = 5.0 × 10−7 M.

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