Abstract:

<p>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 (pNOF), at two unique sites in the protein have been determined. Amber codon-suppression methodology was used to site-specifically incorporate pNOF at a solvent-accessible (Asp133) and a partially buried (Asn149) site in sfGFP. The Asp133pNOF sfGFP construct crystallized with two molecules per asymmetric unit in space group P321 and the crystal structure was refined to 2.05 Å resolution. Crystals of Asn149pNOF sfGFP contained one molecule of sfGFP per asymmetric unit in space group P422 and the structure was refined to 1.60 Å resolution. The alignment of Asp133pNOF or Asn149pNOF 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 pNOF as an effective spectroscopic reporter of local protein structure and dynamics.</p>