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Structure of the red fluorescent protein from a lancelet (Branchiostoma lanceolatum): a novel GYG chromophore covalently bound to a nearby tyrosine.

ABSTRACT: A key property of proteins of the green fluorescent protein (GFP) family is their ability to form a chromophore group by post-translational modifications of internal amino acids, e.g. Ser65-Tyr66-Gly67 in GFP from the jellyfish Aequorea victoria (Cnidaria). Numerous structural studies have demonstrated that the green GFP-like chromophore represents the `core' structure, which can be extended in red-shifted proteins owing to modifications of the protein backbone at the first chromophore-forming position. Here, the three-dimensional structures of green laGFP (?ex/?em = 502/511?nm) and red laRFP (?ex/?em ? 521/592?nm), which are fluorescent proteins (FPs) from the lancelet Branchiostoma lanceolatum (Chordata), were determined together with the structure of a red variant laRFP-?S83 (deletion of Ser83) with improved folding. Lancelet FPs are evolutionarily distant and share only ?20% sequence identity with cnidarian FPs, which have been extensively characterized and widely used as genetically encoded probes. The structure of red-emitting laRFP revealed three exceptional features that have not been observed in wild-type fluorescent proteins from Cnidaria reported to date: (i) an unusual chromophore-forming sequence Gly58-Tyr59-Gly60, (ii) the presence of Gln211 at the position of the conserved catalytic Glu (Glu222 in Aequorea GFP), which proved to be crucial for chromophore formation, and (iii) the absence of modifications typical of known red chromophores and the presence of an extremely unusual covalent bond between the Tyr59?C(?) atom and the hydroxyl of the proximal Tyr62. The impact of this covalent bond on the red emission and the large Stokes shift (?70?nm) of laRFP was verified by extensive structure-based site-directed mutagenesis.

SUBMITTER: Pletnev VZ

PROVIDER: S-EPMC3760133 | biostudies-literature | 2013 Sep

REPOSITORIES: biostudies-literature

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Structure of the red fluorescent protein from a lancelet (Branchiostoma lanceolatum): a novel GYG chromophore covalently bound to a nearby tyrosine.

Pletnev Vladimir Z VZ Pletneva Nadya V NV Lukyanov Konstantin A KA Souslova Ekaterina A EA Fradkov Arkady F AF Chudakov Dmitry M DM Chepurnykh Tatyana T Yampolsky Ilia V IV Wlodawer Alexander A Dauter Zbigniew Z Pletnev Sergei S

Acta crystallographica. Section D, Biological crystallography 20130817 Pt 9

A key property of proteins of the green fluorescent protein (GFP) family is their ability to form a chromophore group by post-translational modifications of internal amino acids, e.g. Ser65-Tyr66-Gly67 in GFP from the jellyfish Aequorea victoria (Cnidaria). Numerous structural studies have demonstrated that the green GFP-like chromophore represents the `core' structure, which can be extended in red-shifted proteins owing to modifications of the protein backbone at the first chromophore-forming p ...[more]

PMID: 23999308

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Project description:BackgroundThe basally divergent phylogenetic position of amphioxus (Cephalochordata), as well as its conserved morphology, development and genetics, make it the best proxy for the chordate ancestor. Particularly, studies using the amphioxus model help our understanding of vertebrate evolution and development. Thus, interest for the amphioxus model led to the characterization of both the transcriptome and complete genome sequence of the American species, Branchiostoma floridae. However, recent technical improvements allowing induction of spawning in the laboratory during the breeding season on a daily basis with the Mediterranean species Branchiostoma lanceolatum have encouraged European Evo-Devo researchers to adopt this species as a model even though no genomic or transcriptomic data have been available. To fill this need we used the pyrosequencing method to characterize the B. lanceolatum transcriptome and then compared our results with the published transcriptome of B. floridae.ResultsStarting with total RNA from nine different developmental stages of B. lanceolatum, a normalized cDNA library was constructed and sequenced on Roche GS FLX (Titanium mode). Around 1.4 million of reads were produced and assembled into 70,530 contigs (average length of 490 bp). Overall 37% of the assembled sequences were annotated by BlastX and their Gene Ontology terms were determined. These results were then compared to genomic and transcriptomic data of B. floridae to assess similarities and specificities of each species.ConclusionWe obtained a high-quality amphioxus (B. lanceolatum) reference transcriptome using a high throughput sequencing approach. We found that 83% of the predicted genes in the B. floridae complete genome sequence are also found in the B. lanceolatum transcriptome, while only 41% were found in the B. floridae transcriptome obtained with traditional Sanger based sequencing. Therefore, given the high degree of sequence conservation between different amphioxus species, this set of ESTs may now be used as the reference transcriptome for the Branchiostoma genus.

Dataset Information

Structure of the red fluorescent protein from a lancelet (Branchiostoma lanceolatum): a novel GYG chromophore covalently bound to a nearby tyrosine.

Publications

Structure of the red fluorescent protein from a lancelet (Branchiostoma lanceolatum): a novel GYG chromophore covalently bound to a nearby tyrosine.

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