Crystal Structure of Enhanced Green Fluorescent Protein to 1.35 Å Resolution Reveals Alternative Conformations for Glu222

Arpino, James A. J.; Rizkallah, P.J.; Jones, D. Dafydd

doi:10.1371/journal.pone.0047132

Cited by 130 publications

(151 citation statements)

References 26 publications

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“…1A). The crystal structure of eGFP (34) and molecular modeling of the structure of NanoLuc were used to guide the design of the chimeric reporter (Fig. 1B).…”

Section: Resultsmentioning

confidence: 99%

Fluorophore-NanoLuc BRET Reporters Enable Sensitive In Vivo Optical Imaging and Flow Cytometry for Monitoring Tumorigenesis

et al. 2015

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Fluorescent proteins are widely used to study molecular and cellular events, yet this traditionally relies on delivery of excitation light, which can trigger autofluorescence, photoxicity, and photobleaching, impairing their use in vivo. Accordingly, chemiluminescent light sources such as those generated by luciferases have emerged, as they do not require excitation light. However, current luciferase reporters lack the brightness needed to visualize events in deep tissues. We report the creation of chimeric eGFP-NanoLuc (GpNLuc) and LSSmOrange-NanoLuc (OgNLuc) fusion reporter proteins coined LumiFluors, which combine the benefits of eGFP or LSSmOrange fluorescent proteins with the bright, glow-type bioluminescent light generated by an enhanced small luciferase subunit (NanoLuc) of the deep sea shrimp Oplophorus gracilirostris. The intramolecular bioluminescence resonance energy transfer (BRET) that occurs between NanoLuc and the fused fluorophore generates the brightest bioluminescent signal known to date, including improved intensity, sensitivity and durable spectral properties, thereby dramatically reducing image acquisition times and permitting highly sensitive in vivo imaging. Notably, the self-illuminating and bi-functional nature of these LumiFluor reporters enables greatly improved spatio-temporal monitoring of very small numbers of tumor cells via in vivo optical imaging and also allows the isolation and analyses of single cells by flow cytometry. Thus, LumiFluor reporters are inexpensive, robust, non-invasive tools that allow for markedly improved in vivo optical imaging of tumorigenic processes.

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“…1A). The crystal structure of eGFP (34) and molecular modeling of the structure of NanoLuc were used to guide the design of the chimeric reporter (Fig. 1B).…”

Section: Resultsmentioning

confidence: 99%

Fluorophore-NanoLuc BRET Reporters Enable Sensitive In Vivo Optical Imaging and Flow Cytometry for Monitoring Tumorigenesis

et al. 2015

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“…Interactions in absorption spectrum of those residues were determined by realizing mutations as it was presented in several previous studies [18,37,38]. Some common empirical rules can be extracted from these results, for instance, mutations of T203V, T203I or T203Y eliminate a hydrogen bond on the chromophore phenolate ring in anionic state and shift to redthe absorption energy [18].…”

Section: Resultsmentioning

confidence: 99%

Fluorescence of a Histidine-Modified Enhanced Green Fluorescent Protein (EGFP) Effectively Quenched by Copper(II) Ions. Part II. Molecular Determinants

et al. 2015

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The histidine-modified EGFP was characterized as a sensing element that preferentially binds nanomolar concentrations of Cu(2+) in a reversible manner (Kd = 15 nM). This research aims to determine the causes of nanomolar-affinity of this mutant by investigating significant structural and energetic alterations of the chromophore in the presence of different copper ion concentrations. In order to reveal the unknown parts of the quenching mechanism we have elaborated a specific approach that combines theoretical and experimental techniques. The theoretical experiment included the modeling of potential distortions of the chromophores and the corresponding changes in energy using quantum mechanical calculations. Differences between the modeled energy profiles of planar and distorted conformations represented the energies of activation for the chromophore distortions. We found that some values of the experimental activation energies, which were derived from fluorescence lifetime decay analysis (ex: 470 nm, em: 507 nm), were consistent with the theoretical ones. Thus, it has been revealed similarity between the theoretical activation energy (50 kJmol(-1)) for 40° phenolate-ring distortion and the experimental activation energy (52.17 kJmol(-1)) required for histidine-modified EGFP saturation with copper. This chromophore conformation was further investigated and it has been found that the large decrease in fluorescence emission is attributed to the significant charge transfer over the molecule which triggers proton transfer thereby neutralizing the cromophore.

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“…rsGreen0.7 shows a similar overall structure as other GFP-like FPs, with a typical 11-stranded barrel fold and a central helix holding the chromophore in place ( Figure S12, supporting information). 38,54 All mutations affecting the photoswitching behavior are located near the chromophore and are oriented inwards. This includes the original rsEGFP mutations (Q69L, V150A, V163S and S205N) 26 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 18 density, here modelled with two conformations.…”

Section: Colimentioning

confidence: 99%

Expression-Enhanced Fluorescent Proteins Based on Enhanced Green Fluorescent Protein for Super-resolution Microscopy

Duwé¹,

Zitter²,

Gielen³

et al. 2015

ACS Nano

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Smart fluorophores", such as reversibly switchable fluorescent proteins (RSFPs), are crucial for advanced fluorescence imaging. However, only a limited number of such labels is available and many display reduced biological performance compared to more classical variants.We present the development of robustly photoswitchable variants of EGFP, named rsGreens, that display up to 30-fold higher fluorescence in E. coli colonies grown at 37°C and more than 4-fold higher fluorescence when expressed in HEK293T cells compared to their ancestor protein rsEGFP. This enhancement is not due to an intrinsic increase in the fluorescence brightness of the probes, but rather due to enhanced expression levels that allow many more probe molecules to be functional at any given time. We developed rsGreens displaying a range of photoswitching kinetics and show how these can be used for multi-modal diffraction-unlimited fluorescence imaging such as pcSOFI and RESOLFT, achieving a spatial resolution of ~70 nm. By determining the first ever crystal structures of a negative reversibly switchable FP derived from Aequorea victoria in both the "on"-and "off"-conformation we were able to confirm the presence of a cis-trans isomerization and provide further insights into the mechanisms underlying the photochromism. Our work demonstrates that genetically encoded "smart fluorophores" can be readily optimized for biological performance, and provides a practical strategy for developing maturation-and stability-enhanced photochromic fluorescent proteins.KEYWORDS: fluorescent proteins, reversible photoswitching, super-resolution fluorescence microscopy, SOFI, RESOLFT, crystal structure determination, rsEGFP, superfolder Fluorescent proteins (FPs) enable the minimally-invasive labeling of intracellular structures in live systems. 1 The discovery and development of "smart photoactive FPs", 2,3 with features such as irreversible photoactivation and photoconversion, or reversible photoswitching, allowed the development of diffraction-unlimited imaging techniques such as (f)PALM 4,5 ((fluorescence) photoactivated localization microscopy), RESOLFT 6 (reversible saturable optical fluorescence transitions) and (pc)SOFI 7,8 ((photochromic) stochastic optical fluctuation imaging). These techniques strongly rely on the performance of the fluorophores and considerable efforts have therefore been dedicated to create optimized "smart labels". 9 This is exemplified by the continuous optimization and diversification of the EosFP family, 10-15 or the development of Dronpa 16 mutants with different or added photophysical properties. [17][18][19][20][21][22] Probes that combine multiple "smart" behaviors have also been engineered. [23][24][25] On the whole, however, the general acceptance of the FP-based "smart labels" has not quite risen up to the high expectations set by the many applications they enable. In some cases this is due to concerns surrounding the biological compatibility of the labels, meaning that the label may interfere with the functioning of the syst...

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Crystal Structure of Enhanced Green Fluorescent Protein to 1.35 Å Resolution Reveals Alternative Conformations for Glu222

Cited by 130 publications

References 26 publications

Fluorophore-NanoLuc BRET Reporters Enable Sensitive In Vivo Optical Imaging and Flow Cytometry for Monitoring Tumorigenesis

Fluorophore-NanoLuc BRET Reporters Enable Sensitive In Vivo Optical Imaging and Flow Cytometry for Monitoring Tumorigenesis

Fluorescence of a Histidine-Modified Enhanced Green Fluorescent Protein (EGFP) Effectively Quenched by Copper(II) Ions. Part II. Molecular Determinants

Expression-Enhanced Fluorescent Proteins Based on Enhanced Green Fluorescent Protein for Super-resolution Microscopy

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