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Title: Room temperature crystal structure of the fast switching M159T mutant of the fluorescent protein dronpa

Authors:
; ; ; ; ; ; ; ;  [1];  [2];  [2]
  1. (Jyvaskyla)
  2. (
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
OTHER
OSTI Identifier:
1170039
Resource Type:
Journal Article
Resource Relation:
Journal Name: Proteins; Journal Volume: 83; Journal Issue: (3) ; 03, 2015
Country of Publication:
United States
Language:
ENGLISH

Citation Formats

Kaucikas, Marius, Fitzpatrick, Ann, Bryan, Elana, Struve, Abelone, Henning, Robert, Kosheleva, Irina, Srajer, Vukica, Groenhof, Gerrit, Van Thor, Jasper J., ICL), and UC). Room temperature crystal structure of the fast switching M159T mutant of the fluorescent protein dronpa. United States: N. p., 2015. Web. doi:10.1002/prot.24742.
Kaucikas, Marius, Fitzpatrick, Ann, Bryan, Elana, Struve, Abelone, Henning, Robert, Kosheleva, Irina, Srajer, Vukica, Groenhof, Gerrit, Van Thor, Jasper J., ICL), & UC). Room temperature crystal structure of the fast switching M159T mutant of the fluorescent protein dronpa. United States. doi:10.1002/prot.24742.
Kaucikas, Marius, Fitzpatrick, Ann, Bryan, Elana, Struve, Abelone, Henning, Robert, Kosheleva, Irina, Srajer, Vukica, Groenhof, Gerrit, Van Thor, Jasper J., ICL), and UC). Sun . "Room temperature crystal structure of the fast switching M159T mutant of the fluorescent protein dronpa". United States. doi:10.1002/prot.24742.
@article{osti_1170039,
title = {Room temperature crystal structure of the fast switching M159T mutant of the fluorescent protein dronpa},
author = {Kaucikas, Marius and Fitzpatrick, Ann and Bryan, Elana and Struve, Abelone and Henning, Robert and Kosheleva, Irina and Srajer, Vukica and Groenhof, Gerrit and Van Thor, Jasper J. and ICL) and UC)},
abstractNote = {},
doi = {10.1002/prot.24742},
journal = {Proteins},
number = (3) ; 03, 2015,
volume = 83,
place = {United States},
year = {Sun Mar 01 00:00:00 EST 2015},
month = {Sun Mar 01 00:00:00 EST 2015}
}
  • No abstract prepared.
  • The fluorescent protein fromDendronephthyasp. (DendFP) is a member of the Kaede-like group of photoconvertible fluorescent proteins with a His62-Tyr63-Gly64 chromophore-forming sequence. Upon irradiation with UV and blue light, the fluorescence of DendFP irreversibly changes from green (506 nm) to red (578 nm). The photoconversion is accompanied by cleavage of the peptide backbone at the C α—N bond of His62 and the formation of a terminal carboxamide group at the preceding Leu61. The resulting double C α=C βbond in His62 extends the conjugation of the chromophore π system to include imidazole, providing the red fluorescence. Here, the three-dimensional structures of nativemore » green and photoconverted red forms of DendFP determined at 1.81 and 2.14 Å resolution, respectively, are reported. This is the first structure of photoconverted red DendFP to be reported to date. The structure-based mutagenesis of DendFP revealed an important role of positions 142 and 193: replacement of the original Ser142 and His193 caused a moderate red shift in the fluorescence and a considerable increase in the photoconversion rate. It was demonstrated that hydrogen bonding of the chromophore to the Gln116 and Ser105 cluster is crucial for variation of the photoconversion rate. The single replacement Gln116Asn disrupts the hydrogen bonding of Gln116 to the chromophore, resulting in a 30-fold decrease in the photoconversion rate, which was partially restored by a further Ser105Asn replacement.« less
  • The red fluorescent protein variant TagRFP-T has greatly improved photostability over its parent molecule, TagRFP, but the underlying mechanism leading to this improvement is to date unknown. The 1.95 Å resolution crystallographic structure of TagRFP-T showed that its chromophore exists as a mixture of cis and trans coplanar isomers in roughly equal proportions. Interestingly, both isomers are able to fluoresce, a property that has never been observed in any other fluorescent protein. We propose a “circular restoration model” for TagRFP-T to explain its superior photostability: There are four co-existing chromophore states (cis/trans protonated/ionized state) that can be driven by light tomore » transform from one state into another. This model also explains how TagRPF-T essentially eliminates the temporary dark state (reversible photobleaching). - Highlights: • The 1.95 Å resolution crystal structure of TagRFP-T was determined. • The chromophore of TagRFP-T contains a mixture of cis and trans coplanar isomers. • A “circular restoration model” was proposed to explain the superior photostability. • The chromophore can reversibly convert between cis/trans protonated/ionized states. • The light-driven conversion reduce the dark state (reversible photobleaching).« less
  • Phanta is a reversibly photoswitching chromoprotein (Φ F, 0.003), useful for pcFRET, that was isolated from a mutagenesis screen of the bright green fluorescent eCGP123 (Φ F, 0.8). We have investigated the contribution of substitutions at positions His193, Thr69 and Gln62, individually and in combination, to the optical properties of Phanta. Single amino acid substitutions at position 193 resulted in proteins with very low Φ F, indicating the importance of this position in controlling the fluorescence efficiency of the variant proteins. The substitution Thr69Val in Phanta was important for supressing the formation of a protonated chromophore species observed in somemore » His193 substituted variants, whereas the substitution Gln62Met did not significantly contribute to the useful optical properties of Phanta. X-ray crystal structures for Phanta (2.3 Å), eCGP123 T69V (2.0 Å) and eCGP123 H193Q (2.2 Å) in their non-photoswitched state were determined, revealing the presence of a cis-coplanar chromophore. We conclude that changes in the hydrogen-bonding network supporting the cis-chromophore, and its contacts with the surrounding protein matrix, are responsible for the low fluorescence emission of eCGP123 variants containing a His193 substitution.« less