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Title: Crystal structure of the fluorescent protein from Dendronephthya sp. in both green and photoconverted red forms

Abstract

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 native 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 ofmore » Gln116 to the chromophore, resulting in a 30-fold decrease in the photoconversion rate, which was partially restored by a further Ser105Asn replacement.« less

Authors:
; ; ; ; ; ; ;
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
USDOE
OSTI Identifier:
1314255
Resource Type:
Journal Article
Resource Relation:
Journal Name: Acta Crystallographica. Section D. Structural Biology; Journal Volume: 72; Journal Issue: 8
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Pletneva, Nadya V., Pletnev, Sergei, Pakhomov, Alexey A., Chertkova, Rita V., Martynov, Vladimir I., Muslinkina, Liya, Dauter, Zbigniew, and Pletnev, Vladimir Z. Crystal structure of the fluorescent protein from Dendronephthya sp. in both green and photoconverted red forms. United States: N. p., 2016. Web. doi:10.1107/S205979831601038X.
Pletneva, Nadya V., Pletnev, Sergei, Pakhomov, Alexey A., Chertkova, Rita V., Martynov, Vladimir I., Muslinkina, Liya, Dauter, Zbigniew, & Pletnev, Vladimir Z. Crystal structure of the fluorescent protein from Dendronephthya sp. in both green and photoconverted red forms. United States. doi:10.1107/S205979831601038X.
Pletneva, Nadya V., Pletnev, Sergei, Pakhomov, Alexey A., Chertkova, Rita V., Martynov, Vladimir I., Muslinkina, Liya, Dauter, Zbigniew, and Pletnev, Vladimir Z. 2016. "Crystal structure of the fluorescent protein from Dendronephthya sp. in both green and photoconverted red forms". United States. doi:10.1107/S205979831601038X.
@article{osti_1314255,
title = {Crystal structure of the fluorescent protein from Dendronephthya sp. in both green and photoconverted red forms},
author = {Pletneva, Nadya V. and Pletnev, Sergei and Pakhomov, Alexey A. and Chertkova, Rita V. and Martynov, Vladimir I. and Muslinkina, Liya and Dauter, Zbigniew and Pletnev, Vladimir Z.},
abstractNote = {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 Cα—N bond of His62 and the formation of a terminal carboxamide group at the preceding Leu61. The resulting double Cα=Cβbond in His62 extends the conjugation of the chromophore π system to include imidazole, providing the red fluorescence. Here, the three-dimensional structures of native 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.},
doi = {10.1107/S205979831601038X},
journal = {Acta Crystallographica. Section D. Structural Biology},
number = 8,
volume = 72,
place = {United States},
year = 2016,
month = 7
}
  • No abstract prepared.
  • In this paper, we describe the engineering and X-ray crystal structure of Thermal Green Protein (TGP), an extremely stable, highly soluble, non-aggregating green fluorescent protein. TGP is a soluble variant of the fluorescent protein eCGP123, which despite being highly stable, has proven to be aggregation-prone. The X-ray crystal structure of eCGP123, also determined within the context of this paper, was used to carry out rational surface engineering to improve its solubility, leading to TGP. The approach involved simultaneously eliminating crystal lattice contacts while increasing the overall negative charge of the protein. Despite intentional disruption of lattice contacts and introduction ofmore » high entropy glutamate side chains, TGP crystallized readily in a number of different conditions and the X-ray crystal structure of TGP was determined to 1.9 Å resolution. The structural reasons for the enhanced stability of TGP and eCGP123 are discussed. We demonstrate the utility of using TGP as a fusion partner in various assays and significantly, in amyloid assays in which the standard fluorescent protein, EGFP, is undesirable because of aberrant oligomerization.« less
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