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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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Decay via twisted internal charge-transfer states of the chromophore with mechanism for coupling to proton transfer.A schematic depiction of the hypothetical photoswitching mechanism. The mechanism is a variation of the photoswtiching mechanism for negative-mode reversibly photo switchable fluorescent proteins suggested by Olsen Lamothe & Martínez [62]. The key point is that there are two twisted intramolecular charge-transfer channels available to the anionic chromophore, whose ground state is a quantum superposition of states with distinct bond alternation. The excited state is the anti-phase combination of the same two bonding states. The two channels correspond to the two bonds on the bridge. The neutral chromophore only has one TICT channel, corresponding to the I bond, reflecting the definite bond alternation of its ground state. The neutral excited state has opposing bond alternation and charge localisation, relative to its ground state. If the anionic chromophore accesses the imidazolinone TICT channel, then its excited state becomes photo basic relative to the optically excited state, increasing the probability that it will accept a proton. Acceptance of a proton by the anion TICT state in the imidazolinone channel deactivates the excited state, yielding a neutral chromophore population in its ground state. If the anion accesses the phenoxy TICT channel in its excited state, electron density is pushed off the phenoxy, making it more photo acidic than the optically prepared excited state, and preventing proton uptake. Deactivation of the anionic excited state can still occur through the conical intersections that exist in the phenoxy TICT channel, yielding an anionic ground state. If the combined rate of access of the phenoxy and imidazolinoxy channels is much larger than the rate of fluorescence, then the "ON" state is non-fluorescent, as observed in Phanta. The kinetics of photoswitching will depend on the precise shape of the electronic state surfaces and on the friction experienced by the chromophore during its excited-state dynamics. The coupling between TICT state channels and protonation state can be understood using an adaptation of a two-state electron transfer model [74].
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pone.0123338.g005: Decay via twisted internal charge-transfer states of the chromophore with mechanism for coupling to proton transfer.A schematic depiction of the hypothetical photoswitching mechanism. The mechanism is a variation of the photoswtiching mechanism for negative-mode reversibly photo switchable fluorescent proteins suggested by Olsen Lamothe & Martínez [62]. The key point is that there are two twisted intramolecular charge-transfer channels available to the anionic chromophore, whose ground state is a quantum superposition of states with distinct bond alternation. The excited state is the anti-phase combination of the same two bonding states. The two channels correspond to the two bonds on the bridge. The neutral chromophore only has one TICT channel, corresponding to the I bond, reflecting the definite bond alternation of its ground state. The neutral excited state has opposing bond alternation and charge localisation, relative to its ground state. If the anionic chromophore accesses the imidazolinone TICT channel, then its excited state becomes photo basic relative to the optically excited state, increasing the probability that it will accept a proton. Acceptance of a proton by the anion TICT state in the imidazolinone channel deactivates the excited state, yielding a neutral chromophore population in its ground state. If the anion accesses the phenoxy TICT channel in its excited state, electron density is pushed off the phenoxy, making it more photo acidic than the optically prepared excited state, and preventing proton uptake. Deactivation of the anionic excited state can still occur through the conical intersections that exist in the phenoxy TICT channel, yielding an anionic ground state. If the combined rate of access of the phenoxy and imidazolinoxy channels is much larger than the rate of fluorescence, then the "ON" state is non-fluorescent, as observed in Phanta. The kinetics of photoswitching will depend on the precise shape of the electronic state surfaces and on the friction experienced by the chromophore during its excited-state dynamics. The coupling between TICT state channels and protonation state can be understood using an adaptation of a two-state electron transfer model [74].

Mentions: Phanta and a number of the eCGP123H193X variants reported in this study (Table 1) are unique amongst the class of reversible photoswitchable fluorescent proteins (RSFP) in that they are weakly fluorescent both in their ON and OFF states. The possibility of photochromic switching without fluorescence in GFP-like proteins was recently predicted in a hypothetical photoswitching mechanism based on the chemistry of the model chromophore p-hydroxybenzylideneimidazolinone (HBI) [62]. The model proposes that coupling between protonation and bond isomerisation state in fluorescent protein chromophores is a natural consequence of the coupling between charge localisation and bond twisting in the excited state of the chromophore itself [62]. The mechanism (outlined Fig 5) helps to rationalise some properties of Phanta.


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)

Decay via twisted internal charge-transfer states of the chromophore with mechanism for coupling to proton transfer.A schematic depiction of the hypothetical photoswitching mechanism. The mechanism is a variation of the photoswtiching mechanism for negative-mode reversibly photo switchable fluorescent proteins suggested by Olsen Lamothe & Martínez [62]. The key point is that there are two twisted intramolecular charge-transfer channels available to the anionic chromophore, whose ground state is a quantum superposition of states with distinct bond alternation. The excited state is the anti-phase combination of the same two bonding states. The two channels correspond to the two bonds on the bridge. The neutral chromophore only has one TICT channel, corresponding to the I bond, reflecting the definite bond alternation of its ground state. The neutral excited state has opposing bond alternation and charge localisation, relative to its ground state. If the anionic chromophore accesses the imidazolinone TICT channel, then its excited state becomes photo basic relative to the optically excited state, increasing the probability that it will accept a proton. Acceptance of a proton by the anion TICT state in the imidazolinone channel deactivates the excited state, yielding a neutral chromophore population in its ground state. If the anion accesses the phenoxy TICT channel in its excited state, electron density is pushed off the phenoxy, making it more photo acidic than the optically prepared excited state, and preventing proton uptake. Deactivation of the anionic excited state can still occur through the conical intersections that exist in the phenoxy TICT channel, yielding an anionic ground state. If the combined rate of access of the phenoxy and imidazolinoxy channels is much larger than the rate of fluorescence, then the "ON" state is non-fluorescent, as observed in Phanta. The kinetics of photoswitching will depend on the precise shape of the electronic state surfaces and on the friction experienced by the chromophore during its excited-state dynamics. The coupling between TICT state channels and protonation state can be understood using an adaptation of a two-state electron transfer model [74].
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4414407&req=5

pone.0123338.g005: Decay via twisted internal charge-transfer states of the chromophore with mechanism for coupling to proton transfer.A schematic depiction of the hypothetical photoswitching mechanism. The mechanism is a variation of the photoswtiching mechanism for negative-mode reversibly photo switchable fluorescent proteins suggested by Olsen Lamothe & Martínez [62]. The key point is that there are two twisted intramolecular charge-transfer channels available to the anionic chromophore, whose ground state is a quantum superposition of states with distinct bond alternation. The excited state is the anti-phase combination of the same two bonding states. The two channels correspond to the two bonds on the bridge. The neutral chromophore only has one TICT channel, corresponding to the I bond, reflecting the definite bond alternation of its ground state. The neutral excited state has opposing bond alternation and charge localisation, relative to its ground state. If the anionic chromophore accesses the imidazolinone TICT channel, then its excited state becomes photo basic relative to the optically excited state, increasing the probability that it will accept a proton. Acceptance of a proton by the anion TICT state in the imidazolinone channel deactivates the excited state, yielding a neutral chromophore population in its ground state. If the anion accesses the phenoxy TICT channel in its excited state, electron density is pushed off the phenoxy, making it more photo acidic than the optically prepared excited state, and preventing proton uptake. Deactivation of the anionic excited state can still occur through the conical intersections that exist in the phenoxy TICT channel, yielding an anionic ground state. If the combined rate of access of the phenoxy and imidazolinoxy channels is much larger than the rate of fluorescence, then the "ON" state is non-fluorescent, as observed in Phanta. The kinetics of photoswitching will depend on the precise shape of the electronic state surfaces and on the friction experienced by the chromophore during its excited-state dynamics. The coupling between TICT state channels and protonation state can be understood using an adaptation of a two-state electron transfer model [74].
Mentions: Phanta and a number of the eCGP123H193X variants reported in this study (Table 1) are unique amongst the class of reversible photoswitchable fluorescent proteins (RSFP) in that they are weakly fluorescent both in their ON and OFF states. The possibility of photochromic switching without fluorescence in GFP-like proteins was recently predicted in a hypothetical photoswitching mechanism based on the chemistry of the model chromophore p-hydroxybenzylideneimidazolinone (HBI) [62]. The model proposes that coupling between protonation and bond isomerisation state in fluorescent protein chromophores is a natural consequence of the coupling between charge localisation and bond twisting in the excited state of the chromophore itself [62]. The mechanism (outlined Fig 5) helps to rationalise some properties of Phanta.

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.

Show MeSH
Related in: MedlinePlus