1. Crystal structures of green fluorescent protein with the unnatural amino acid 4-nitro-L-phenylalanine.
- Author
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Maurici N, Savidge N, Lee BU, Brewer SH, and Phillips-Piro CM
- Subjects
- Alanine chemistry, Alanine metabolism, Amino Acid Sequence, Amino Acid Substitution, Asparagine metabolism, Aspartic Acid metabolism, Binding Sites, Cloning, Molecular, Crystallography, X-Ray, Escherichia coli genetics, Escherichia coli metabolism, Gene Expression, Genes, Reporter, Genetic Vectors chemistry, Genetic Vectors metabolism, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Models, Molecular, Mutagenesis, Site-Directed, Nitriles metabolism, Phenylalanine chemistry, Phenylalanine metabolism, Protein Binding, Protein Conformation, alpha-Helical, Protein Conformation, beta-Strand, Protein Engineering methods, Protein Interaction Domains and Motifs, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Structural Homology, Protein, Alanine analogs & derivatives, Asparagine chemistry, Aspartic Acid chemistry, Green Fluorescent Proteins chemistry, Nitriles chemistry, Phenylalanine analogs & derivatives
- 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 (pNO
2 F), at two unique sites in the protein have been determined. Amber codon-suppression methodology was used to site-specifically incorporate pNO2 F at a solvent-accessible (Asp133) and a partially buried (Asn149) site in sfGFP. The Asp133pNO2 F sfGFP construct crystallized with two molecules per asymmetric unit in space group P32 21 and the crystal structure was refined to 2.05 Å resolution. Crystals of Asn149pNO2 F sfGFP contained one molecule of sfGFP per asymmetric unit in space group P41 22 and the structure was refined to 1.60 Å resolution. The alignment of Asp133pNO2 F or Asn149pNO2 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 pNO2 F as an effective spectroscopic reporter of local protein structure and dynamics.- Published
- 2018
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