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.

Source: Acta Crystallographica Section F - September 19, 2018 Category: Biochemistry Authors: Maurici, N. Savidge, N. Lee, B.U. Brewer, S.H. Phillips-Piro, C.M. Tags: 4-nitro-l-phenylalanine unnatural amino acids noncanonical amino acids green fluorescent protein research communications Source Type: research