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

Craig B Don Paul, Daouda A K Traore, Seth Olsen, Rodney J Devenish, Devin W Close, Toby D M Bell, Andrew Bradbury, Matthew C J Wilce, Mark Prescott

Research output: Contribution to journalArticleResearchpeer-review

Abstract

Phanta is a reversibly photoswitching chromoprotein (PhiF, 0.003), useful for pcFRET, that was isolated from a mutagenesis screen of the bright green fluorescent eCGP123 (PhiF, 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 PhiF, 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 A), eCGP123T69V (2.0 A) and eCGP123H193Q (2.2 A) 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.
Original languageEnglish
Article numbere0123338
Number of pages23
JournalPLoS ONE
Volume10
Issue number4
DOIs
Publication statusPublished - 2015

Cite this

@article{62b4dd72770c4832963b1173b8be08c8,
title = "X-ray crystal structure and properties of phanta, a weakly fluorescent photochromic GFP-like protein",
abstract = "Phanta is a reversibly photoswitching chromoprotein (PhiF, 0.003), useful for pcFRET, that was isolated from a mutagenesis screen of the bright green fluorescent eCGP123 (PhiF, 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 PhiF, 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 A), eCGP123T69V (2.0 A) and eCGP123H193Q (2.2 A) 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.",
author = "{Don Paul}, {Craig B} and Traore, {Daouda A K} and Seth Olsen and Devenish, {Rodney J} and Close, {Devin W} and Bell, {Toby D M} and Andrew Bradbury and Wilce, {Matthew C J} and Mark Prescott",
year = "2015",
doi = "10.1371/journal.pone.0123338",
language = "English",
volume = "10",
journal = "PLoS ONE",
issn = "1932-6203",
publisher = "Public Library of Science",
number = "4",

}

X-ray crystal structure and properties of phanta, a weakly fluorescent photochromic GFP-like protein. / Don Paul, Craig B; Traore, Daouda A K; Olsen, Seth; Devenish, Rodney J; Close, Devin W; Bell, Toby D M; Bradbury, Andrew; Wilce, Matthew C J; Prescott, Mark.

In: PLoS ONE, Vol. 10, No. 4, e0123338, 2015.

Research output: Contribution to journalArticleResearchpeer-review

TY - JOUR

T1 - X-ray crystal structure and properties of phanta, a weakly fluorescent photochromic GFP-like protein

AU - Don Paul, Craig B

AU - Traore, Daouda A K

AU - Olsen, Seth

AU - Devenish, Rodney J

AU - Close, Devin W

AU - Bell, Toby D M

AU - Bradbury, Andrew

AU - Wilce, Matthew C J

AU - Prescott, Mark

PY - 2015

Y1 - 2015

N2 - Phanta is a reversibly photoswitching chromoprotein (PhiF, 0.003), useful for pcFRET, that was isolated from a mutagenesis screen of the bright green fluorescent eCGP123 (PhiF, 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 PhiF, 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 A), eCGP123T69V (2.0 A) and eCGP123H193Q (2.2 A) 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.

AB - Phanta is a reversibly photoswitching chromoprotein (PhiF, 0.003), useful for pcFRET, that was isolated from a mutagenesis screen of the bright green fluorescent eCGP123 (PhiF, 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 PhiF, 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 A), eCGP123T69V (2.0 A) and eCGP123H193Q (2.2 A) 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.

UR - http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4414407/pdf/pone.0123338.pdf

U2 - 10.1371/journal.pone.0123338

DO - 10.1371/journal.pone.0123338

M3 - Article

VL - 10

JO - PLoS ONE

JF - PLoS ONE

SN - 1932-6203

IS - 4

M1 - e0123338

ER -