The fluorescence of the chromophore of the green fluorescent protein of Aequorea and Renilla

McCapra, Frank; Razavi, Zia; Neary, Adrian P.

doi:10.1039/c39880000790

Cited by 29 publications

(16 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The fluorescence has been attributed to the presence of a chromophore in the protein, formed by the cyclization of three amino acid residues within an hexapeptide segment of the polypeptide chain by post-translational modification [8,10]. Considerable work has gone into the chemical structure of the chromophore [8,10,21], but the chemical basis for the fluorescence is still unknown. During the purification of His-GFP, a marked increase in fluorescence intensity was observed when the harvested cells and cell-precipitate were allowed to stand overnight at room temperature.…”

Section: Resultsmentioning

confidence: 99%

“…It is possible that the photobleaching may be due to a reduction taking place within the cell and the recovery of fluorescence to the transfer of electrons to molecular oxygen. The proposed imidazolone structure for the chromophore of GFP [8,10,21], however, does not appear to explain the redox behavior of rGFP in that the electron acceptor~zlonor group is not identifiable. Another interesting structure-related problem is that Aequorea GFP, when expressed in such diverse organisms as E. coli, C. elegans and COS cells, is fluorescent [13,20].…”

Section: Electronmentioning

confidence: 99%

See 1 more Smart Citation

Evidence for redox forms of the Aequorea green fluorescent protein

1994

View full text Add to dashboard Cite

Highly purified recombinant Aequorea green fluorescent protein is able to undergo a reversible oxidation-reduction reaction in the presence of molecular oxygen. In the oxidized form in near UV light, the protein is highly fluorescent, but when reduced with sodium dithionite, it becomes completely non-fluorescent. On exposure to molecular oxygen the reduced, non-fluorescent protein reverts to its original fluorescent state.

show abstract

Section: Resultsmentioning

confidence: 99%

Section: Electronmentioning

confidence: 99%

Evidence for redox forms of the Aequorea green fluorescent protein

1994

View full text Add to dashboard Cite

show abstract

“…analog of 6e with an ethylenic double bond linked to the 2-position of the imidazolidinone was synthesized by McCapra et al (16) and measured to absorb at 445 nm. In general, ZINDO more closely reproduced the experimental wavelengths than did TDHDFT.…”

Section: Resultsmentioning

confidence: 99%

The structure of the chromophore within DsRed, a red fluorescent protein from coral

Gross

Baird

Hoffman

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

578

594

View full text Add to dashboard Cite

DsRed, a brilliantly red fluorescent protein, was recently cloned from Discosoma coral by homology to the green fluorescent protein (GFP) from the jellyfish Aequorea. A core question in the biochemistry of DsRed is the mechanism by which the GFP-like 475-nm excitation and 500-nm emission maxima of immature DsRed are red-shifted to the 558-nm excitation and 583-nm emission maxima of mature DsRed. After digestion of mature DsRed with lysyl endopeptidase, high-resolution mass spectra of the purified chromophore-bearing peptide reveal that some of the molecules have lost 2 Da relative to the peptide analogously prepared from a mutant, K83R, that stays green. Tandem mass spectrometry indicates that the bond between the alpha-carbon and nitrogen of Gln-66 has been dehydrogenated in DsRed, extending the GFP chromophore by forming OCANOCAO at the 2-position of the imidazolidinone. This acylimine substituent quantitatively accounts for the red shift according to quantum mechanical calculations. Reversible hydration of the CAN bond in the acylimine would explain why denaturation shifts mature DsRed back to a GFP-like absorbance. The CAN bond hydrolyses upon boiling, explaining why DsRed shows two fragment bands on SDS͞PAGE. This assay suggests that conversion from green to red chromophores remains incomplete even after prolonged aging. In the preceding companion paper (1), we showed that the red fluorescent protein DsRed cloned from coral by Matz et al. (2) has impressive brightness and stability against pH changes, denaturants, and photobleaching, and that it can be mutated to even longer wavelengths of excitation and emission. However, DsRed also shows slow and complex kinetics of maturation, proceeding via a green fluorescent protein (GFP)-like green intermediate to the final red species. DsRed also proves to be at least an obligate tetramer, two of which may weakly associate into an octamer (1). The present paper examines one of the most important fundamental unsolved questions about DsRed, namely the structure of the red chromophore and the mechanism by which it attains such long wavelengths. Matz et al. (2) speculated that ''an additional autocatalytic reaction, presumably inhibited in GFP, takes place during fluorophore maturation and leads to the extension of its conjugated -system.'' In an accompanying commentary, Tsien (3) raised the possibility that an amine side chain might attack the terminal carbonyl of the GFP chromophore to form an iminium cation somewhat analogous to that in rhodopsin. More recently, Terskikh et al.** suggested instead that ''the additional reaction is another cyclization within the backbone rather than attachment of a group derived from a neighboring side chain.'' All of these suggestions have been of generic reaction types rather than specific proposals of a detailed structure for the red chromophore.Our strategy relied critically on a point mutant, K83R, which remains almost entirely arrested at the green stage of maturation (1) and provides a reference standard for that intermediate...

show abstract

“…When the absorption and fluorescence spectra of native and renatured native GFP are compared at neutral pH, the spectra are found to be identical, indicating that the denaturation of GFP is reversible and that the fluorescence is dependent on the protein environment (16). However, a model compound of the chromophore has been found to be nonfluorescent in a variety of solvents (17).…”

mentioning

confidence: 99%

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

Niwa

Inouye

Hirano

et al. 1996

Proc. Natl. Acad. Sci. U.S.A.

434

442

View full text Add to dashboard Cite

The jellyfish Aequorea victoria possesses in the margin of its umbrella a green f luorescent 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 f luorescence ( max ‫؍‬ 508 nm) when irradiated with long ultraviolet light. The f luorescence is due to the presence of a chromophore consisting of an imidazolone ring, formed by a post-translational modification of the tripeptide -Ser 65 -Tyr 66 -Gly 67 -. 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 f luorescence 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.

show abstract

The fluorescence of the chromophore of the green fluorescent protein of Aequorea and Renilla

Cited by 29 publications

References 0 publications

Evidence for redox forms of the Aequorea green fluorescent protein

Evidence for redox forms of the Aequorea green fluorescent protein

The structure of the chromophore within DsRed, a red fluorescent protein from coral

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

Contact Info

Product

Resources

About