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X-Ray Crystal Structure and Properties of Phanta, a Weakly Fluorescent Photochromic GFP-Like Protein.

Don Paul C, Traore DA, Olsen S, Devenish RJ, Close DW, Bell TD, Bradbury A, Wilce MC, Prescott M - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuro- and Sensory Physiology, University Medicine, Göttingen, 37073, Göttingen, Germany.

ABSTRACT
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.

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Optical spectra of Phanta and selected variants.(A) Spectra determined at pH 8.0 are shown for Phanta and variants of eCGP123 containing amino acid substitutions that contribute to Phanta. Absorbance (solid line), fluorescence excitation (dashed line) and fluorescence emission (dotted line) are shown. (B) The absorbance spectra are shown for selected variants at pH 8.0 (solid line), pH 6.0 (dashed line) and pH 3.0 (dotted line). (C) Absorbance spectra determined at pH 8.0 are shown for variants of eCGP123 singly substituted at position 193 or doubly substituted at positions 193 and 69.
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pone.0123338.g001: Optical spectra of Phanta and selected variants.(A) Spectra determined at pH 8.0 are shown for Phanta and variants of eCGP123 containing amino acid substitutions that contribute to Phanta. Absorbance (solid line), fluorescence excitation (dashed line) and fluorescence emission (dotted line) are shown. (B) The absorbance spectra are shown for selected variants at pH 8.0 (solid line), pH 6.0 (dashed line) and pH 3.0 (dotted line). (C) Absorbance spectra determined at pH 8.0 are shown for variants of eCGP123 singly substituted at position 193 or doubly substituted at positions 193 and 69.

Mentions: The singly substituted variants, eCGP123H193Q eCGP123T69V, eCGP123Q62M were expressed with a C-terminal 6 x His tag to facilitate protein purification. The absorbance and fluorescence excitation and emission properties were determined at pH 8.0 for each of the purified non-photoswitched proteins (Fig 1 and Table 1). Compared to the parent eCGP123 (λmaxabs, 495 nm), the absorbance spectrum for Phanta (λmaxabs, 506 nm) is significantly red-shifted by 11 nm, resulting in increased spectral overlap with the EGFP emission spectrum [5]. The absorbance spectrum of eCGP123T69V showed a major absorbing species at 506 nm whilst eCGP123H193Q contained significant amounts of a 387 nm species in addition to the 506 nm species. We estimated the proportion anionic and protonated chromophore species in eCGP123H193Q using the molar absorbance extinction coefficients for the fully anionic chomphore of Phanta, and the fully protonated chromophore of eCGP123H19D of eCGP123H193E. The fraction of protonated to anionic chromphore CGP123H193Q at pH8.0 was estimated to be 2.4 to 1 suggesting that the majority chromophore species is the protonated state.


X-Ray Crystal Structure and Properties of Phanta, a Weakly Fluorescent Photochromic GFP-Like Protein.

Don Paul C, Traore DA, Olsen S, Devenish RJ, Close DW, Bell TD, Bradbury A, Wilce MC, Prescott M - PLoS ONE (2015)

Optical spectra of Phanta and selected variants.(A) Spectra determined at pH 8.0 are shown for Phanta and variants of eCGP123 containing amino acid substitutions that contribute to Phanta. Absorbance (solid line), fluorescence excitation (dashed line) and fluorescence emission (dotted line) are shown. (B) The absorbance spectra are shown for selected variants at pH 8.0 (solid line), pH 6.0 (dashed line) and pH 3.0 (dotted line). (C) Absorbance spectra determined at pH 8.0 are shown for variants of eCGP123 singly substituted at position 193 or doubly substituted at positions 193 and 69.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4414407&req=5

pone.0123338.g001: Optical spectra of Phanta and selected variants.(A) Spectra determined at pH 8.0 are shown for Phanta and variants of eCGP123 containing amino acid substitutions that contribute to Phanta. Absorbance (solid line), fluorescence excitation (dashed line) and fluorescence emission (dotted line) are shown. (B) The absorbance spectra are shown for selected variants at pH 8.0 (solid line), pH 6.0 (dashed line) and pH 3.0 (dotted line). (C) Absorbance spectra determined at pH 8.0 are shown for variants of eCGP123 singly substituted at position 193 or doubly substituted at positions 193 and 69.
Mentions: The singly substituted variants, eCGP123H193Q eCGP123T69V, eCGP123Q62M were expressed with a C-terminal 6 x His tag to facilitate protein purification. The absorbance and fluorescence excitation and emission properties were determined at pH 8.0 for each of the purified non-photoswitched proteins (Fig 1 and Table 1). Compared to the parent eCGP123 (λmaxabs, 495 nm), the absorbance spectrum for Phanta (λmaxabs, 506 nm) is significantly red-shifted by 11 nm, resulting in increased spectral overlap with the EGFP emission spectrum [5]. The absorbance spectrum of eCGP123T69V showed a major absorbing species at 506 nm whilst eCGP123H193Q contained significant amounts of a 387 nm species in addition to the 506 nm species. We estimated the proportion anionic and protonated chromophore species in eCGP123H193Q using the molar absorbance extinction coefficients for the fully anionic chomphore of Phanta, and the fully protonated chromophore of eCGP123H19D of eCGP123H193E. The fraction of protonated to anionic chromphore CGP123H193Q at pH8.0 was estimated to be 2.4 to 1 suggesting that the majority chromophore species is the protonated state.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neuro- and Sensory Physiology, University Medicine, Göttingen, 37073, Göttingen, Germany.

ABSTRACT
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.

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