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

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.