A Structural Basis for the pH-dependent Increase in Fluorescence Efficiency of Chromoproteins

Battad, Jion; Wilmann, P.G.; Olsen, Seth; Byres, E.; Smith, Sean C.; Dove, Sophie; Turcic, Kristina; Devenish, Rodney J.; Rossjohn, Jamie; Prescott, Mark

doi:10.1016/j.jmb.2007.02.007

Cited by 27 publications

(59 citation statements)

References 68 publications

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“…The X-ray crystal structure of Rtms5 suggests that its low fluorescence emission results from the trans non-coplanar configuration of the chromophore derived from an Gln-Tyr-Gly tripeptide [15]. An Rtms5 H146S variant was significantly more fluorescent than Rtms5 particularly at high pH (Φ F , 0.16 at pH 11.0; , 630 nm), and the X-ray crystal structure showed evidence for a chromophore in a cis -coplanar configuration [16]. The chromophore in Rtms5 is extended by the presence of an acylimine linkage, and is in part responsible for the red-shifted optical properties of this protein [15]–[18].…”

Section: Introductionmentioning

confidence: 97%

A Green Fluorescent Protein Containing a QFG Tri-Peptide Chromophore: Optical Properties and X-Ray Crystal Structure

et al. 2012

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Rtms5 is an deep blue weakly fluorescent GFP-like protein (, 592 nm; , 630nm; ΦF, 0.004) that contains a 66Gln-Tyr-Gly chromophore tripeptide sequence. We investigated the optical properties and structure of two variants, Rtms5Y67F and Rtms5Y67F/H146S in which the tyrosine at position 67 was substituted by a phenylalanine. Compared to the parent proteins the optical spectra for these new variants were significantly blue-shifted. Rtms5Y67F spectra were characterised by two absorbing species (, 440 nm and 513 nm) and green fluorescence emission (, 440 nm; , 508 nm; ΦF, 0.11), whilst Rtms5Y67F/H146S spectra were characterised by a single absorbing species (, 440 nm) and a relatively high fluorescence quantum yield (ΦF, 0.75; , 440 nm; , 508 nm). The fluorescence emissions of each variant were remarkably stable over a wide range of pH (3–11). These are the first GFP-like proteins with green emissions (500–520 nm) that do not have a tyrosine at position 67. The X-ray crystal structure of each protein was determined to 2.2 Å resolution and showed that the benzylidine ring of the chromophore, similar to the 4-hydroxybenzylidine ring of the Rtms5 parent, is non-coplanar and in the trans conformation. The results of chemical quantum calculations together with the structural data suggested that the 513 nm absorbing species in Rtms5Y67F results from an unusual form of the chromophore protonated at the acylimine oxygen. These are the first X-ray crystal structures for fluorescent proteins with a functional chromophore containing a phenylalanine at position 67.

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Section: Introductionmentioning

confidence: 97%

A Green Fluorescent Protein Containing a QFG Tri-Peptide Chromophore: Optical Properties and X-Ray Crystal Structure

et al. 2012

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“…The excited-state twisting processes of the chromophores of GFP variants are of direct technological interest because of their implication in binding-dependent fluorescence enhancement, 55 reversible photoconversion processes, [56][57][58][59] and light-activated assembly of split-protein constructs. 60,61 In this paper, we describe a simple two-state model Hamiltonian that can describe the potential energy and the charge-transfer dependence of monomethine dyes at different detuning from the cyanine limit.…”

Section: Introductionmentioning

confidence: 99%

A two-state model of twisted intramolecular charge-transfer in monomethine dyes

Olsen

McKenzie

2012

The Journal of Chemical Physics

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Articles you may be interested inModeling opto-electronic properties of a dye molecule in proximity of a semiconductor nanoparticle J. Chem. Phys. 139, 024105 (2013) A two-state model Hamiltonian is proposed, which can describe the coupling of twisting displacements to charge-transfer behavior in the ground and excited states of a general monomethine dye molecule. This coupling may be relevant to the molecular mechanism of environment-dependent fluorescence yield enhancement. The model is parameterized against quantum chemical calculations on different protonation states of the green fluorescent protein chromophore, which are chosen to sample different regimes of detuning from the cyanine (resonant) limit. The model provides a simple yet realistic description of the charge transfer character along two possible excited state twisting channels associated with the methine bridge. It describes qualitatively different behavior in three regions that can be classified by their relationship to the resonant (cyanine) limit. The regimes differ by the presence or absence of twist-dependent polarization reversal and the occurrence of conical intersections. We find that selective biasing of one twisting channel over another by an applied diabatic biasing potential can only be achieved in a finite range of parameters near the cyanine limit.

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“…The recent determination of structure of the RFP HcRed [57] -one of only a handful of RFPs that have been structurally characterized to date -was achieved by a combination of X-ray structure and optical spectroscopy aided by our quantum chemical modeling. More recently, a remarkable enhancement in fluorescence efficiency at high pH of the weakly fluorescent RFP Rtms5 has been reported, structurally characterized and mechanistically rationalized by the same collaborative team [60]. The mechanistic picture that emerges hinges on the protonation state of the chromophore and its surrounding residues, which not only impacts the electronic structure, and consequently the absorption and emission wavelengths, but also the structural stability of its more highly fluorescent isomeric forms.…”

Section: Internal Conversion Mechanism In Fp Chromophoresmentioning

confidence: 95%

“…This has led to a series of publications [51][52][53][54][56][57][58] that have elaborated both key structural and mechanistic issues relating to the RFP HcRed [57] and pocilloporin Rtms5 H146S [59,60] in recent years, and provides an important experimental context within which the knowledge gained from our calculations can aid in furthering experimental development of new engineered proteins. In this regard simulations and modeling can add fundamental understanding to the direction aspect of a directed evolution approach, since the physical interactions and mechanisms introduced by mutations that yield improved RFPs are unknown, and mutagenesis studies alone cannot reveal them.…”

Section: Introductionmentioning

confidence: 99%

Theoretical Studies of Green and Red Fluorescent Proteins

Zhang

Sun

Wang

et al. 2010

Hydrogen Bonding and Transfer in the Excited State

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The green fluorescent protein (GFP) of the Aequorea victoria jellyfish (and its structural analogues, e.g., red fluorescent protein) has emerged as a unique fluorescent label and has evolved into an extraordinarily important platform for biotechnological and cell biology applications due to its amazing ability to generate a highly visible, efficiently emitting internal fluorophore (for an overview, see review articles [1,2]). Its unique photophysical properties are nowadays being commonly exploited for imaging studies of protein folding, gene expression, protein trafficking and cell development, since the gene in GFP contains all the information necessary for the post-translational synthesis of the chromophore and expression of the gene in other organisms can create fluorescence. Structurally, GFP is an 11-stranded b-barrel, which is nearly a perfect cylinder, threaded by an a-helix running up the axis of the cylinder (Figure 36.1) [3,4]. The chromophore is attached to the a-helix and is buried almost perfectly in the centre of the cylinder. The chromophore (p-hydroxybenzylideneimidazolinone) is auto-catalytically generated by the post-translational modification of a three-amino-acid sequence. This characteristic structure of GFP can both constrain the motions of the chromophore and shield it from the surrounding water solvent that would otherwise quench its fluorescence. A surprising number of polar groups and structured water molecules are buried adjacent to the chromophore and appear to play a crucial role in the functionality of the system through their participation in hydrogen bonding network. Owing to the importance of GFP as a bio-imaging reagent, there is substantial interest in understanding the photophysics of the embedded chromophore and in particular in determining the structural changes and the dynamics in and around the chromophore following the photo-excitation. Various

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A Structural Basis for the pH-dependent Increase in Fluorescence Efficiency of Chromoproteins

Cited by 27 publications

References 68 publications

A Green Fluorescent Protein Containing a QFG Tri-Peptide Chromophore: Optical Properties and X-Ray Crystal Structure

A Green Fluorescent Protein Containing a QFG Tri-Peptide Chromophore: Optical Properties and X-Ray Crystal Structure

A two-state model of twisted intramolecular charge-transfer in monomethine dyes

Theoretical Studies of Green and Red Fluorescent Proteins

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