A New Strategy to Photoactivate Green Fluorescent Protein

Groff, Dan; Wang, Feng; Jockusch, Steffen; Turro, Nicholas J.; Schultz, Peter G.

doi:10.1002/anie.201003797

Cited by 37 publications

(34 citation statements)

References 23 publications

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“…To our great surprise, the sfGFP66ONBY structure revealed a domain-swapped dimeric arrangement with residues 1-143 (domain 1) Clear electron density identified the connecting loops ranging from residues Tyr143 to His148 (Fig. 3C), while no electron density was present in the position corresponding to b-strand b 7 in the search model (as previously observed by Groff et al [23]). The electron density extends away from the search model, leading to the second half-subunit where the protein backbone is also shifted due to the steric interference of the bulky ortho-nitrobenzyl-group with the normal tight packing of the b 7 -strand against the chromophore.…”

Section: Crystal Structure Of Dark-state Sfgfp66onby Shows (Dimeric) supporting

confidence: 80%

Crystal structure of a domain‐swapped photoactivatable sfGFP variant provides evidence for GFP folding pathway

et al. 2019

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Photoactivatable fluorescent proteins (PA‐FPs) are a powerful non‐invasive tool in high‐resolution live‐cell imaging. They can be converted from an inactive to an active form by light, enabling the spatial and temporal trafficking of proteins and cell dynamics. PA‐FPs have been previously generated by mutating selected residues in the chromophore or in its close proximity. A new strategy to generate PA‐FPs is the genetic incorporation of unnatural amino acids (UAAs) containing photocaged groups using unique suppressor tRNA/aminoacyl‐tRNA synthetase pairs. We set out to develop a photoactivatable GFP variant suitable for time‐resolved structural studies. Here, we report the crystal structure of superfolder GFP (sfGFP) containing the UAA ortho‐nitrobenzyl‐tyrosine (ONBY) at position 66 and its spectroscopic characterization. Surprisingly, the crystal structure (to 2.7 Å resolution) reveals a dimeric domain‐swapped arrangement of sfGFP66ONBY with residues 1–142 of one molecule associating with residues 148–234 from another molecule. This unusual domain‐swapped structure supports a previously postulated GFP folding pathway that proceeds via an equilibrium intermediate.

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Section: Crystal Structure Of Dark-state Sfgfp66onby Shows (Dimeric) supporting

confidence: 80%

Crystal structure of a domain‐swapped photoactivatable sfGFP variant provides evidence for GFP folding pathway

et al. 2019

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“…519 The relative brightness at the wavelength of maximum emission (486 nm) increased by at least 4 orders of magnitude during photoactivation at 365 nm.…”

Section: Photoactivatable Fluorescent Dyesmentioning

confidence: 98%

Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy

et al. 2012

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“…To improve their overall performance in abottomup manner,n umerouse xperimental and theoretical studies have been dedicated to investigating the underlying photophysicala nd photochemical mechanism of natural GFPs and chemically synthesized GFP core chromophores. [30][31][32][33][34][35][36][37][38][39] In contrastt oG FP chromophores, the photophysics and photochemistry of the blue fluorescenceprotein (BFP) chromophore and its variants are rarely explored. Earlier studies [40][41][42][43][44][45] have primarily focused on spectroscopic properties.…”

Section: Introductionmentioning

confidence: 99%

Excited‐State Intramolecular Proton Transfer in a Blue Fluorescence Chromophore Induces Dual Emission

Guo

et al. 2016

ChemPhysChem

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Compared with green fluorescence protein (GFP) chromophores, the recently synthesized blue fluorescence protein (BFP) chromophore variant presents intriguing photochemical properties, for example, dual fluorescence emission, enhanced fluorescence quantum yield, and ultra-slow excited-state intramolecular proton transfer (ESIPT; J. Phys. Chem. Lett., 2014, 5, 92); however, its photochemical mechanism is still elusive. Herein we have employed the CASSCF and CASPT2 methods to study the mechanistic photochemistry of a truncated BFP chromophore variant in the S0 and S1 states. Based on the optimized minima, conical intersections, and minimum-energy paths (ESIPT, photoisomerization, and deactivation), we have found that the system has two competitive S1 relaxation pathways from the Franck-Condon point of the BFP chromophore variant. One is the ESIPT path to generate an S1 tautomer that exhibits a large Stokes shift in experiments. The generated S1 tautomer can further evolve toward the nearby S1 /S0 conical intersection and then jumps down to the S0 state. The other is the photoisomerization path along the rotation of the central double bond. Along this path, the S1 system runs into an S1 /S0 conical intersection region and eventually hops to the S0 state. The two energetically allowed S1 excited-state deactivation pathways are responsible for the in-part loss of fluorescence quantum yield. The considerable S1 ESIPT barrier and the sizable barriers that separate the S1 tautomers from the S1 /S0 conical intersections make these two tautomers establish a kinetic equilibrium in the S1 state, which thus results in dual fluorescence emission.

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A New Strategy to Photoactivate Green Fluorescent Protein

Cited by 37 publications

References 23 publications

Crystal structure of a domain‐swapped photoactivatable sfGFP variant provides evidence for GFP folding pathway

Crystal structure of a domain‐swapped photoactivatable sfGFP variant provides evidence for GFP folding pathway

Photoremovable Protecting Groups in Chemistry and Biology: Reaction Mechanisms and Efficacy

Excited‐State Intramolecular Proton Transfer in a Blue Fluorescence Chromophore Induces Dual Emission

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