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

ABSTRACT: 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 (pNO2F), at two unique sites in the protein have been determined. Amber codon-suppression methodology was used to site-specifically incorporate pNO2F at a solvent-accessible (Asp133) and a partially buried (Asn149) site in sfGFP. The Asp133pNO2F sfGFP construct crystallized with two molecules per asymmetric unit in space group P3221 and the crystal structure was refined to 2.05?Å resolution. Crystals of Asn149pNO2F sfGFP contained one molecule of sfGFP per asymmetric unit in space group P4122 and the structure was refined to 1.60?Å resolution. The alignment of Asp133pNO2F or Asn149pNO2F 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 pNO2F as an effective spectroscopic reporter of local protein structure and dynamics.

SUBMITTER: Maurici N

PROVIDER: S-EPMC6168768 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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

Maurici Nicole N Savidge Nicole N Lee Byung Uk BU Brewer Scott H SH Phillips-Piro Christine M CM

Acta crystallographica. Section F, Structural biology communications 20180919 Pt 10

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 (pNO2F), at two unique sites in the protein have been determined. Amber codon-suppression methodology was used to site-specifically incorporate pNO2F at a solvent-accessible (Asp133) and a partially buried (Asn149) site in sfGFP. The Asp133pNO2F sfGFP construct cryst ...[more]

PMID: 30279317

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Project description:The crystal structures of two ammonium salts of 2-amino-4-nitro-benzoic acid are described, namely di-methyl-aza-nium 2-amino-4-nitro-benzoate, C2H8N+·C7H5N2O4-, (I), and di-butyl-aza-nium 2-amino-4-nitro-benzoate, C8H20N+·C7H5N2O4-, (II). The asymmetric unit of (I) comprises a single cation and a single anion. In the anion, small twists are noted for the carboxyl-ate and nitro groups from the ring to which they are connected, as indicated by the dihedral angles of 11.45 (13) and 3.71 (15)°, respectively; the dihedral angle between the substituents is 7.9 (2)°. The asymmetric unit of (II) comprises two independent pairs of cations and anions. In the cations, different conformations are noted in the side chains in that three chains have an all-trans [(+)-anti-periplanar] conformation, while one has a distinctive kink resulting in a (+)-synclinal conformation. The anions, again, exhibit twists with the dihedral angles between the carboxyl-ate and nitro groups and the ring being 12.73 (6) and 4.30 (10)°, respectively, for the first anion and 8.1 (4) and 12.6 (3)°, respectively, for the second. The difference between anions in (I) and (II) is that in the anions of (II), the terminal groups are conrotatory, forming dihedral angles of 17.02 (8) and 19.0 (5)°, respectively. In each independent anion of (I) and (II), an intra-molecular amino-N-H⋯O(carboxyl-ate) hydrogen bond is formed. In the crystal of (I), anions are linked into a jagged supra-molecular chain by charge-assisted amine-N-H⋯O(carboxyl-ate) hydrogen bonds and these are connected into layers via charge-assisted ammonium-N-H⋯O(carboxyl-ate) hydrogen bonds. The resulting layers stack along the a axis, being connected by nitro-N-O⋯π(arene) and methyl-C-H⋯O(nitro) inter-actions. In the crystal of (II), the anions are connected into four-ion aggregates by charge-assisted amino-N-H⋯O(carboxyl-ate) hydrogen bonding. The formation of ammonium-N-H⋯O(carboxyl-ate) hydrogen bonds, involving all ammonium-N-H and carboxyl-ate O atoms leads to a three-dimensional architecture; additional C-H⋯O(nitro) inter-actions contribute to the packing. The Hirshfeld surface analysis confirms the importance of the hydrogen bonding in both crystal structures. Indeed, O⋯H/H⋯O inter-actions contribute nearly 50% to the entire Hirshfeld surface in (I).

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Dataset Information

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

Publications

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

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