Molecular basis for functional switching of GFP by two disparate non-native post-translational modifications of a phenyl azide reaction handle

Hartley, Andrew M.; Worthy, Harley L.; Reddington, Samuel C.; Rizkallah, P.J.; Jones, D. Dafydd

doi:10.1039/c6sc00944a

Cited by 20 publications

(31 citation statements)

References 52 publications

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“…His148 is dynamic 41 and has been observed in both the "open" and "closed" conformations with the former not normally reported in the crystal structure as it is a minor component when observed (Figure 2a and S3) that generates a channel through to the chromophore (Figure 6b-c). Such a tunnel at a similar position has been observed previously for GFP-like proteins 46,47 . Thus, His148 may acts as a "gatekeeper" residue, so determining access to the chromophore as well as its functional role 41 .…”

Section: Oxygen Access To the Chromophore And The Twisted Gly67supporting

confidence: 85%

“…Photolysis experiments were carried out using a UVM-57 Handheld UV lamp (6 W; 302 nm UV, UVP Cambridge, UK) and 1 cm pathlength quartz cuvette (Hellma), essentially as described previously 29,46 . A maximum of 500 μl of protein sample (10 μM) was pipetted into a cuvette and exposed to the UV (302 nm) for the indicated periods of time at a distance of 1cm.…”

Section: Photolysis Of Venus 66azfmentioning

confidence: 99%

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Stalling chromophore maturation of the fluorescent protein Venus reveals the molecular basis of the final oxidation step

Auhim

Grigorenko

Harris

et al. 2020

Preprint

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Fluorescent proteins (FPs) have revolutionised the life sciences but the mechanism of chromophore maturation is still not fully understood. Incorporation of a photo-responsive non-canonical amino acid within the chromophore stalls maturation of Venus, a yellow FP, at an intermediate stage; the crystal structure reveals the presence of O2 located above a dehydrated enolate imidazolone (I) ring, close to the strictly conserved Gly67 that occupies a twisted conformation. His148 adopts an open conformation, potentially allowing O2 access to the chromophore. Absorption spectroscopy supported by QM/MM simulations suggest that the first oxidation step involves formation of a hydroperoxyl intermediate in conjunction with dehydrogenation of the methylene bridge. A fully conjugated mature chromophore is formed through release of H2O2 upon irradiation of this intermediate, both in vitro and in vivo. The possibility of interrupting and photochemically restarting chromophore maturation, and the mechanistic insights opens up new approaches for engineering optically controlled fluorescent proteins.

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Section: Oxygen Access To the Chromophore And The Twisted Gly67supporting

confidence: 85%

Section: Photolysis Of Venus 66azfmentioning

confidence: 99%

Stalling chromophore maturation of the fluorescent protein Venus reveals the molecular basis of the final oxidation step

Auhim

Grigorenko

Harris

et al. 2020

Preprint

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“…Residue His148 plays a crucial role in the deprotonation of Tyr66 [63]. Mutation of H148 to a ncAA results in the breakage of this key H-bond causing the CRO A chromophore to predominate [51,54]. The formation of sfGFP homodimers using SPAAC compatible residues at 148 not only reverses this protonation state so switching on CRO B, but enhances brightness over threefold above wild type sfGFP — indicative of functional synergy [62].…”

Section: Use Of Non-canonical Amino Acidsmentioning

confidence: 99%

Better together: building protein oligomers naturally and by design

Gwyther

Jones

Worthy

2019

Biochemical Society Transactions

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Protein oligomers are more common in nature than monomers, with dimers being the most prevalent final structural state observed in known structures. From a biological perspective, this makes sense as it conserves vital molecular resources that may be wasted simply by generating larger single polypeptide units, and allows new features such as cooperativity to emerge. Taking inspiration from nature, protein designers and engineers are now building artificial oligomeric complexes using a variety of approaches to generate new and useful supramolecular protein structures. Oligomerisation is thus offering a new approach to sample structure and function space not accessible through simply tinkering with monomeric proteins.

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“…Spectroscopic reportercontaining UAAs represent one subclass of UAAs that can be utilized to study these features in proteins. UAAs containing vibrational reporters, fluorescent groups and NMR probes have all been genetically incorporated into protein systems (Schultz et al, 2006;Jackson et al, 2007;Cellitti et al, 2008;Miyake-Stoner et al, 2010;Ye et al, 2010;Smith et al, 2011;Thielges et al, 2011;Bazewicz et al, 2012Bazewicz et al, , 2013Adhikary et al, 2014;Ma et al, 2015;Tookmanian, Phillips-Piro et al, 2015;Dippel et al, 2016;Hartley et al, 2016;Slocum & Webb, 2016). In order to be effective probes of protein structure, the probes must have both a specific spectroscopic signal sensitive to the local environment and be minimally perturbative to the local protein environment under investigation.…”

Section: Introductionmentioning

confidence: 99%

Crystal structures of green fluorescent protein with the unnatural amino acid 4-nitro-L-phenylalanine

et al. 2018

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The X-ray crystal structures of two superfolder green fluorescent protein (sfGFP) constructs containing a genetically incorporated spectroscopic reporter unnatural amino acid, 4-nitro-l-phenylalanine (pNO 2 F), at two unique sites in the protein have been determined. Amber codon-suppression methodology was used to site-specifically incorporate pNO 2 F at a solvent-accessible (Asp133) and a partially buried (Asn149) site in sfGFP. The Asp133pNO 2 F sfGFP construct crystallized with two molecules per asymmetric unit in space group P3 2 21 and the crystal structure was refined to 2.05 Å resolution. Crystals of Asn149pNO 2 F sfGFP contained one molecule of sfGFP per asymmetric unit in space group P4 1 22 and the structure was refined to 1.60 Å resolution. The alignment of Asp133pNO 2 F or Asn149pNO 2 F sfGFP with wild-type sfGFP resulted in small root-mean-square deviations, illustrating that these residues do not significantly alter the protein structure and supporting the use of pNO 2 F as an effective spectroscopic reporter of local protein structure and dynamics.

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Molecular basis for functional switching of GFP by two disparate non-native post-translational modifications of a phenyl azide reaction handle

Cited by 20 publications

References 52 publications

Stalling chromophore maturation of the fluorescent protein Venus reveals the molecular basis of the final oxidation step

Stalling chromophore maturation of the fluorescent protein Venus reveals the molecular basis of the final oxidation step

Better together: building protein oligomers naturally and by design

Crystal structures of green fluorescent protein with the unnatural amino acid 4-nitro-L-phenylalanine

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