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

Abstract

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...

Photophysics of DsRed, a Red Fluorescent Protein, from the Ensemble to the Single-Molecule Level

“…From SDS-PAGE analysis after a second cation exchange chromatography step, it was estimated that the purity of the samples was better than 90%, but the absorbance ratio remained unchanged. Also, three bands were always encountered in denaturing electrophoresis gels which correspond to protein monomers and to hydrolysis fragments consistent with Gross et al 7 The addition of the N-terminal poly-His tag to DsRed did not affect the absorption spectrum of the protein appreciably, but by accelerating the purification, it became possible to examine the protein sooner after cell lysis. In these early stages, the absorption shoulder at 482 nm appears as a better resolved feature along with a weaker band at 408 nm.…”

“…Both features are associated with an immature chromophore. 7 With time (∼8-12 h after cell lysis), these features diminished or disappeared altogether as the chromophore reached maturity. The features associated with immature chromophores were absent in the samples used for the single-molecule experiments.…”

A monomeric red fluorescent protein with low cytotoxicity

“…Proteins were alkali-denatured with an equal volume of 2 M NaOH and absorbance spectra were measured immediately. It is known that alkali-denatured DsRed-like chromophore converts to the GFP-like one 20 , with an extinction coefficient 44,000 M À 1 cm À 1 at 452 nm under these conditions. On the basis of the absorption of the native and alkalidenatured proteins, molar extinction coefficients for the native states were calculated.…”

Reporter systems forin vivotracking of lactic acid bacteria in animal model studies

“…Brightness of the monomeric mRuby RFP was increased 5 -8-fold by codonoptimization of the marker protein for expression in mammalian cells. 58,59 Furthermore, the usefulness of fluorescent proteins for imaging is significantly influenced by the total number of functional molecules expressed, the percentage of proteins that develop into mature chromophores, the turnover rate of nascent polypeptides into functional chromophores and the time required before photobleaching occurs. 34,38 Phototoxicity is one of the most adverse side effects FPs may have on living cells and tissue.…”