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Title: X-ray crystal structure and properties of Phanta, a weakly fluorescent photochromic GFP-like protein

Abstract

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 some 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.

Authors:
 [1];  [2];  [3];  [2];  [4];  [2];  [4];  [2];  [2]
  1. Univ. Medicine, Gottingen (Germany)
  2. Monash Univ., Victoria (Australia)
  3. The Univ. of Queensland, Queensland (Australia)
  4. Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
Publication Date:
Research Org.:
Los Alamos National Lab. (LANL), Los Alamos, NM (United States)
Sponsoring Org.:
USDOE National Nuclear Security Administration (NNSA)
OSTI Identifier:
1188714
Grant/Contract Number:  
AC52-06NA25396
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
PLoS ONE
Additional Journal Information:
Journal Volume: 10; Journal Issue: 4; Journal ID: ISSN 1932-6203
Publisher:
Public Library of Science
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; Phanta; photochromic; GFP; protein; crystal structure

Citation Formats

Paul, Craig Don, Traore, Daouda A. K., Olsen, Seth, Devenish, Rodney J., Close, Devin W., Bell, Toby D. M., Bradbury, Andrew, Wilce, Matthew C. J., and Prescott, Mark. X-ray crystal structure and properties of Phanta, a weakly fluorescent photochromic GFP-like protein. United States: N. p., 2015. Web. doi:10.1371/journal.pone.0123338.
Paul, Craig Don, Traore, Daouda A. K., Olsen, Seth, Devenish, Rodney J., Close, Devin W., Bell, Toby D. M., Bradbury, Andrew, Wilce, Matthew C. J., & Prescott, Mark. X-ray crystal structure and properties of Phanta, a weakly fluorescent photochromic GFP-like protein. United States. doi:10.1371/journal.pone.0123338.
Paul, Craig Don, Traore, Daouda A. K., Olsen, Seth, Devenish, Rodney J., Close, Devin W., Bell, Toby D. M., Bradbury, Andrew, Wilce, Matthew C. J., and Prescott, Mark. Wed . "X-ray crystal structure and properties of Phanta, a weakly fluorescent photochromic GFP-like protein". United States. doi:10.1371/journal.pone.0123338. https://www.osti.gov/servlets/purl/1188714.
@article{osti_1188714,
title = {X-ray crystal structure and properties of Phanta, a weakly fluorescent photochromic GFP-like protein},
author = {Paul, Craig Don and Traore, Daouda A. K. and Olsen, Seth and Devenish, Rodney J. and Close, Devin W. and Bell, Toby D. M. and Bradbury, Andrew and Wilce, Matthew C. J. and Prescott, Mark},
abstractNote = {Phanta is a reversibly photoswitching chromoprotein (ΦF, 0.003), useful for pcFRET, that was isolated from a mutagenesis screen of the bright green fluorescent eCGP123 (ΦF, 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 ΦF, 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 some 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), eCGP123T69V (2.0 Å) and eCGP123H193Q (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.},
doi = {10.1371/journal.pone.0123338},
journal = {PLoS ONE},
number = 4,
volume = 10,
place = {United States},
year = {Wed Apr 29 00:00:00 EDT 2015},
month = {Wed Apr 29 00:00:00 EDT 2015}
}

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Works referenced in this record:

The Green Fluorescent Protein
journal, June 1998


The creation of a novel fluorescent protein by guided consensus engineering
journal, January 2007

  • Dai, Mingha; Fisher, Hugh E.; Temirov, Jamshid
  • Protein Engineering, Design and Selection, Vol. 20, Issue 2, p. 69-79
  • DOI: 10.1093/protein/gzl056

GFP-like chromoproteins as a source of far-red fluorescent proteins
journal, September 2001