Observation of Excited-State Proton Transfer in Green Fluorescent Protein using Ultrafast Vibrational Spectroscopy

Stoner-Ma, Deborah; Jaye, Andrew A.; Matousek, Pavel; Towrie, Michael; Meech, Stephen R.; Tonge, Peter J.

doi:10.1021/ja042466d

Cited by 187 publications

(266 citation statements)

References 21 publications

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“…The 1721 cm Ϫ1 frequency, characteristic for CϭO stretching of a strongly hydrogen-bonded neutral carboxylic acid (32), and its 11 cm Ϫ1 downshift with H/D exchange suggest Glu-222 as the terminal proton acceptor, because other acidic residues are exposed to solvent and are expected to be fully ionized in equilibrium at pH 8. This evidence is in agreement with a recent proposal (9,11). Further corroboration results from transient infrared measurements on the E222D mutant (11).…”

supporting

confidence: 93%

“…The mechanism by which the protein environment suppresses non-radiative processes remains unexplained. Proposals for the ESPT pathway include a hydrogen bonding network connecting the chromophore phenolic oxygen to Glu-222 via a water molecule and Ser-205 (7)(8)(9). Recently, the possibility of a proton transfer pathway including Glu-222, Asp-82, and Glu-5 was put forward (10).…”

mentioning

confidence: 99%

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Ultrafast and Low Barrier Motions in the Photoreactions of the Green Fluorescent Protein

Thor

Georgiev

Towrie

et al. 2005

Journal of Biological Chemistry

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Green fluorescent protein (GFP) fluoresces efficiently under blue excitation despite major electrostatic rearrangements resulting from photoionization of the chromophore and neutralization of Glu-222. A competing phototransformation process, which ionizes the chromophore and decarboxylates Glu-222, mimics the electrostatic and structural changes in the fluorescence photocycle. Structural and spectroscopic analysis of the cryogenically stabilized photoproduct at 100 K and a structurally annealed intermediate of the phototransformed protein at 170 K reveals distinct structural relaxations involving protein, chromophore, solvent, and photogenerated CO 2 . Strong structural changes of the 100 K photoproduct after decarboxylation appear exclusively within 15 Å of the chromophore and include the electrostatically driven perturbations of Gln-69, Cys-70, and water molecules in an H-bonding network connecting the chromophore. X-ray crystallography to 1.85 Å resolution and static and picosecond time-resolved IR spectroscopy identify structural mechanisms common to phototransformation and to the fluorescence photocycle. In particular, the appearance of a 1697 cm ؊1 (؉) difference band in both photocycle and phototransformation intermediates is a spectroscopic signature for the structural perturbation of Gln-69. This is taken as evidence for an electrostatically driven dynamic response that is common to both photoreaction pathways. The interactions between the chromophore and the perturbed residues and solvent are decreased or removed in the T203H single and T203H/Q69L double mutants, resulting in a strong reduction of the fluorescence quantum yield. This suggests that the electrostatic response to the transient formation of a buried charge in the wild type is important for the bright fluorescence.Green fluorescent protein (GFP) 2 (1, 2) from Aequorea victoria is highly fluorescent with 400 nm excitation, despite major electrostatic rearrangements resulting from rapid charge transfer to the excited chromophore (3). Rapid excited state proton transfer (ESPT) (4 -6) follows excitation of the neutral, phenolic chromophore, and the resulting excited phenolate state I* exhibits high quantum efficiency redshifted emission at 508 nm, with a 3.0-ns lifetime (3). The mechanism by which the protein environment suppresses non-radiative processes remains unexplained. Proposals for the ESPT pathway include a hydrogen bonding network connecting the chromophore phenolic oxygen to Glu-222 via a water molecule and Ser-205 (7-9). Recently, the possibility of a proton transfer pathway including Glu-222, Asp-82, and Glu-5 was put forward (10). The transient infrared absorption reportedly developing at 1706 cm Ϫ1 with excitation of GFP in D 2 O (9) could not distinguish between these proposals. However, experimental evidence for the identity of the proton acceptor has recently been provided from comparison with the E222D mutant (11). Low quantum yield electron transfer from Glu-222 to the photoexcited chromophore triggers decarboxylation of the...

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confidence: 93%

mentioning

confidence: 99%

Ultrafast and Low Barrier Motions in the Photoreactions of the Green Fluorescent Protein

Thor

Georgiev

Towrie

et al. 2005

Journal of Biological Chemistry

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“…This is in sharp contrast to the case in wtGFP, where deuteration dramatically slows the proton-transfer rate. 15,16,19,27 These data suggest that there is a negligible barrier to proton transfer in the excited electronic state. The fast relaxation component reflects the dynamics of the spectral narrowing observed in the gated spectra ( Figure 5).…”

Section: Resultsmentioning

confidence: 91%

“…While proton transfer in wtGFP occurs along a H-bond network, culminating with the protonation of E222, 19 proton transfer in S65T/H148D and E222Q/H148D instead occurs directly from the chromophore to D148. This is made possible by the pre-existence of a low-barrier or barrierless H-bond between the phenol group of the chromophore and the side chain of aspartate.…”

Section: Discussionmentioning

confidence: 99%

An Alternate Proton Acceptor for Excited-State Proton Transfer in Green Fluorescent Protein: Rewiring GFP

et al. 2008

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Abstract:The neutral form of the chromophore in wild-type green fluorescent protein (wtGFP) undergoes excited-state proton transfer (ESPT) upon excitation, resulting in characteristic green (508 nm) fluorescence. This ESPT reaction involves a proton relay from the phenol hydroxyl of the chromophore to the ionized side chain of E222, and results in formation of the anionic chromophore in a protein environment optimized for the neutral species (the I* state). Reorientation or replacement of E222, as occurs in the S65T and E222Q GFP mutants, disables the ESPT reaction and results in loss of green emission following excitation of the neutral chromophore. Previously, it has been shown that the introduction of a second mutation (H148D) into S65T GFP allows the recovery of green emission, implying that ESPT is again possible. A similar recovery of green fluorescence is also observed for the E222Q/H148D mutant, suggesting that D148 is the proton acceptor for the ESPT reaction in both double mutants. The mechanism of fluorescence emission following excitation of the neutral chromophore in S65T/H148D and E222Q/H148D has been explored through the use of steady state and ultrafast time-resolved fluorescence and vibrational spectroscopy. The data are contrasted with those of the single mutant S65T GFP. Time-resolved fluorescence studies indicate very rapid (<1 ps) formation of I* in the double mutants, followed by vibrational cooling on the picosecond time scale. The time-resolved IR difference spectra are markedly different to those of wtGFP or its anionic mutants. In particular, no spectral signatures are apparent in the picosecond IR difference spectra that would correspond to alteration in the ionization state of D148, leading to the proposal that a low-barrier hydrogen bond (LBHB) is present between the phenol hydroxyl of the chromophore and the side chain of D148, with different potential energy surfaces for the ground and excited states. This model is consistent with recent high-resolution structural data in which the distance between the donor and acceptor oxygen atoms is e2.4 Å. Importantly, these studies indicate that the hydrogen-bond network in wtGFP can be replaced by a single residue, an observation which, when fully explored, will add to our understanding of the various requirements for proton-transfer reactions within proteins.

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“…Upon excitation in the blue, the A form of the chromophore loses the phenolic proton converting to an intermediate anionic I form, which in turn infrequently transforms into the thermodynamically stable B form [100]. The neutral chromophore is known to transfer its proton to Glu222 through the wire CRO-WatSer205-Glu222 [101,102] but other differences in the surroundings are difficult to assess experimentally: Since the predominant form of wild-type GFP at room temperature is the neutral one, the structure of the anionic form cannot be established through X-ray diffraction. As workaround, one can adopt as starting model a suitably modified X-ray structure of the mutant S65T which stabilizes the anionic form as also done in earlier computational work [9,103].…”

Section: Resultsmentioning

confidence: 99%

Cutting the Gordian knot of excited-state modeling in complex environments

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Observation of Excited-State Proton Transfer in Green Fluorescent Protein using Ultrafast Vibrational Spectroscopy

Cited by 187 publications

References 21 publications

Ultrafast and Low Barrier Motions in the Photoreactions of the Green Fluorescent Protein

Ultrafast and Low Barrier Motions in the Photoreactions of the Green Fluorescent Protein

An Alternate Proton Acceptor for Excited-State Proton Transfer in Green Fluorescent Protein: Rewiring GFP

Cutting the Gordian knot of excited-state modeling in complex environments

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