Chemical structure of the hexapeptide chromophore of the Aequorea green-fluorescent protein

Cody, Chris W.; Prasher, Douglas C.; Westler, William M.; Prendergast, Franklyn G.; Ward, William W.

doi:10.1021/bi00056a003

Cited by 617 publications

(396 citation statements)

References 12 publications

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“…The origins of the optical properties of GFP, as well as many other fluorescent proteins, can be traced to a chromophore enclosed deep within the β-barrel of the body of the protein. 6,7 In GFP, the chromophore is essentially identical to the deprotonated anion of para-hydroxybenzilidene-2,3-dimethylimidazolinone (HBDI -, shown inset in Figure 1f) and has been widely employed as a model to investigate the intrinsic photophysics of the chromophore within the protein. [8][9][10][11][12][13][14][15][16][17][18] In the gas-phase, the S 1 state is wellcharacterised: the S 1 ← S 0 absorption (action) spectrum is similar to that of the protein and its origin is vertically bound relative to the ground state of the neutral (D 0 ).…”

Section: Introductionmentioning

confidence: 99%

Excited State Dynamics of the Isolated Green Fluorescent Protein Chromophore Anion Following UV Excitation

et al. 2015

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. (2015) 'Excited state dynamics of the isolated green uorescent protein chromophore anion following UV excitation.', Journal of physical chemistry B., 119 (10). pp. 3982-3987. Further information on publisher's website:http://dx.doi.org/10.1021/acs.jpcb.5b01432Publisher's copyright statement:This document is the Accepted Manuscript version of a Published Work that appeared in nal form in The Journal of Physical Chemistry B, copyright c American Chemical Society after peer review and technical editing by the publisher. To access the nal edited and published work see http://dx.doi.org/10.1021/acs.jpcb.5b01432. Additional information:Use policyThe full-text may be used and/or reproduced, and given to third parties in any format or medium, without prior permission or charge, for personal research or study, educational, or not-for-pro t purposes provided that:• a full bibliographic reference is made to the original source • a link is made to the metadata record in DRO • the full-text is not changed in any way The full-text must not be sold in any format or medium without the formal permission of the copyright holders.Please consult the full DRO policy for further details.

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Section: Introductionmentioning

confidence: 99%

Excited State Dynamics of the Isolated Green Fluorescent Protein Chromophore Anion Following UV Excitation

et al. 2015

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“…Its X-ray barrel-type structure was resolved at 1.85Å (PDB entry: 1W7S; [6]), and is formed by eleven β-sheets and one α-helix, to which the GFP chromophore is connected. The latter is obtained by a posttranslational cyclization reaction of the polypeptide skeleton Ser65, Tyr66 and Gly67 residues, followed by oxidation of the Tyr66 residue lateral side-chain [7][8][9]. Through a hydrogen bonds network involving particular polar residues and H 2 O molecules, the cromophore is able to establish noncovalent interactions with the protein [10,11].…”

Section: Introductionmentioning

confidence: 99%

Thermal Effect on Aequorea Green Fluorescent Protein Anionic and Neutral Chromophore Forms Fluorescence

Santos

2011

J Fluoresc

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The emission behaviour of Aequorea green fluorescent protein (A-GFP) chromophore, in both neutral (N) and anionic (A) form, was studied in the temperature range from 20°C to 75°C and at pH=7. Excitation wavelengths of 399 nm and 476 nm were applied to probe the N and A forms environment, respectively. Both forms exhibit distinct fluorescence patterns at high temperature values. The emission quenching rate, following a temperature increase, is higher for the chromophore N form as a result of the hydrogen bond network weakening. The chromophore anionic form emission maximum is red shifted, upon temperature increase, due to a charge transfer process occurring after A form excitation.

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“…One such system is the green fluorescent protein (GFP), a commonly used tool in cellular and molecular imaging owing to the presence of an intrinsically fluorescent chromophore formed autocatalytically from residues S65, Y66, and G67. [10][11][12][13] Alterations in the structure of residues in and around the chromophore perturb the photophysics of GFP, allowing a detailed correlation to be established between chromophore structure and fluorescence. In wild-type GFP (wtGFP), excitation into either the neutral (λ max 395 nm, A) or anionic (λ max 475 nm, B) forms of the chromophore results in very efficient green (λ em 508 and 504 nm, respectively) fluorescence.…”

Section: Introductionmentioning

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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Chemical structure of the hexapeptide chromophore of the Aequorea green-fluorescent protein

Cited by 617 publications

References 12 publications

Excited State Dynamics of the Isolated Green Fluorescent Protein Chromophore Anion Following UV Excitation

Excited State Dynamics of the Isolated Green Fluorescent Protein Chromophore Anion Following UV Excitation

Thermal Effect on Aequorea Green Fluorescent Protein Anionic and Neutral Chromophore Forms Fluorescence

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

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