Structural basis for dual excitation and photoisomerization of the
            <i>Aequorea victoria</i>
            green fluorescent protein

Brejc, Katjuša; Sixma, Titia K.; Kitts, Paul; Kain, Steven R.; Tsien, Roger Y.; Ormö, Mats; Remington, S.J.

doi:10.1073/pnas.94.6.2306

Cited by 692 publications

(948 citation statements)

References 35 publications

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“…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]. From a structural point of view, the protein is highly regular although an exception to this overall behaviour can be observed between strands 7 and 8 of the β-barrel [12,13], whose termini is formed by small sections of the α-helix that resemble "lids".…”

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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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 mutation of S65 to a threonine alters contacts between the side chain of this residue and E222, causing the side chain of the latter to move so that it can no longer participate in ESPT. 18,21 Thus, for S65T, the I* state cannot form, and excitation into A results in only weak fluorescence directly from A*.…”

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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“…Both the A* decay and I* rise are slowed upon deuteration of exchangeable protons, which originally led to the suggestion that the process connecting these states is ultrafast excited-state proton transfer (ESPT) (19,20). The hydrogen bond network near the chromophore observed in the crystal structure of wild-type GFP is suggested to be responsible for ESPT (21). Although this scheme can account for many properties of wild-type GFP photophysics, it is surely incomplete (22)(23)(24)(25)(26).…”

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confidence: 99%

Green Fluorescent Protein Variants as Ratiometric Dual Emission pH Sensors. 2. Excited-State Dynamics

et al. 2002

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], novel mutants of the green fluorescent protein (GFP) that exhibit dual steady-state emission properties were characterized structurally and discussed as potential intracellular pH probes. In this work, the excited-state dynamics of one of these new dual emission GFP variants, deGFP4 (C48S/ S65T/H148C/T203C), is studied by ultrafast fluorescence upconversion spectroscopy. Following excitation of the high-energy absorption band centered at 398 nm and assigned to the neutral form of the chromophore, time-resolved emission was monitored from the excited state of both the neutral and intermediate anionic chromophores at both high and low pH and upon deuteration of exchangeable protons. The time-resolved emission dynamics and isotope effect appear to be very different from those of wild-type GFP [Chattoraj, M., King, B. A., Bublitz, G. U., and Boxer, S. G. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 8362-8367]; however, due to overlapping emission bands, the apparent difference can be analyzed quantitatively within the same framework used to describe GFP excited-state dynamics. The results indicate that the pH-sensitive steady-state emission characteristics of deGFP4 are a result of a pH-dependent modulation of the rate of excited-state proton transfer. At high pH, a rapid interconversion from the excited state of the higher energy neutral chromophore to the lower energy intermediate anionic chromophore is achieved by proton transfer. At low pH, excited-state proton transfer is slowed to the point where it is no longer rate limiting.The green fluorescent protein (GFP) 1 from the jellyfish Aequorea Victoria has become a ubiquitous tool in cell and molecular biology (1). The chromophore of wild-type GFP is formed spontaneously from the internal cyclization and oxidation of the Ser65-Tyr66-Gly67 tripeptide unit (2, 3) and is protectively housed along a coaxial helix threaded through the center of an 11-stranded -barrel (4). The surrounding protein environment is responsible for many of the emission properties of GFP such as the emission wavelength, relative contributions from neutral and anionic forms of the chromophores, and constraints that are responsible for the high fluorescence quantum yield. There has been a significant effort to change the surrounding environment by both random and semirational mutagenesis to produce GFP variants with desirable properties such as new emission wavelengths and improved quantum yields (5, 6), more rapid chromophore maturation (7), greater structural stability (8, 9), and altered sensitivity to environmental properties such as pH (10-12), redox potential (13), and ion concentration (14-16). In the preceding companion paper, a new class of pH-sensitive dual emission GFP (deGFP) mutants is reported (17). These mutants exhibit emission that changes from green (λ max ) 514 nm) to blue (λ max ) 465 nm) as the pH is lowered (Figure 1) and should be useful ratiometric pH sensors in vitro and in vivo. Structural studies of a related dual emission GFP, deGFP1 (S65T/H148G/T203C), show that wh...

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Structural basis for dual excitation and photoisomerization of the Aequorea victoria green fluorescent protein

Cited by 692 publications

References 35 publications

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

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

Green Fluorescent Protein Variants as Ratiometric Dual Emission pH Sensors. 2. Excited-State Dynamics

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