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 (pNO 2 F), at two unique sites in the protein have been determined. Amber codon-suppression methodology was used to site-specifically incorporate pNO 2 F at a solvent-accessible (Asp133) and a partially buried (Asn149) site in sfGFP. The Asp133pNO 2 F sfGFP construct crystallized with two molecules per asymmetric unit in space group P3 2 21 and the crystal structure was refined to 2.05 Å resolution. Crystals of Asn149pNO 2 F sfGFP contained one molecule of sfGFP per asymmetric unit in space group P4 1 22 and the structure was refined to 1.60 Å resolution. The alignment of Asp133pNO 2 F or Asn149pNO 2 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 pNO 2 F as an effective spectroscopic reporter of local protein structure and dynamics.

Structural and spectrophotometric investigation of two unnatural amino-acid altered chromophores in the superfolder green fluorescent protein

“…Visual global comparison of these structures illustrated little change to the -barrel and suggested that most variation in C orientation between the mutant and wild-type structures occurred in a loop region between residues 188 and 195 (Fig. 6a, bottom), similar to what we have observed previously with different sfGFP mutants (Tookmanian et al, 2015;Dippel et al, 2016;Maurici et al, 2018). Examination of the aligned chromophores and neighboring residues in the three structures indicated minimal perturbation in atomic placements upon the incorporation of either pNO 2 Phe or pCNPhe at site 66 (Figs.…”

“…The co-transformed cells were used to inoculate 5 ml non-inducing medium overnight, shaking at 250 rev min À1 at 37 C. Aliquots (2.5 ml) of the cultured cells were used to inoculate 250 ml autoinduction medium (Hammill et al, 2007) containing 1.1 mM pNO 2 Phe or pCNPhe. The cultures were grown with shaking at 250 rev min À1 at 37 C. The cells were harvested after 24-30 h by centrifugation, and the expressed sfGFP protein constructs containing either pNO 2 Phe or pCNPhe were purified by TALON cobalt ion-exchange chromatography as described previously (Dippel et al, 2016;Maurici et al, 2018;Bazewicz et al, 2012).…”

“…Several vibrational reporter UAAs such as 4-nitro-l-phenylalanine (pNO 2 Phe; Fig. 1b; Smith et al, 2011;Maurici et al, 2018) and 4-cyano-lphenylalanine (pCNPhe; Fig. 1c; Bazewicz et al, 2012;Dippel et al, 2016;Slocum & Webb, 2016;First et al, 2018;Slocum et al, 2017) have also been incorporated at various sites in sfGFP to explore differences in local solvation in the protein using IR spectroscopy.…”

Effect of Protein Surface Charge Distribution on Protein–Polyelectrolyte Complexation

“…To assess the effect of different charge patch sizes on protein interactions, sfGFP (PDB ID: 2B3P) was chosen as a model protein because the protein is a robust folder more resistant to denaturation than many proteins, has a known crystal structure, and has known plasticity at many AA positions that allow reconstruction of the protein surface. , In this study, sfGFP protein was mutated to create a panel of mutants with differing electrostatic patch sizes on their surfaces. Mutation sites were selected such that they avoided alterations to structural features (beta barrel and alpha helix) of the proteins, and sites where mutations had previously been reported were preferred. , Selected AAs were mutated to be either positively (Lys and Arg) or negatively charged (Asp and Glu) to create or enlarge charge patches on sfGFP and mutated to be neutral (but not hydrophobic) to decrease or remove charge patches.…”

“…Protein Design, Patch Calculation, and Expression. To assess the effect of different charge patch sizes on protein interactions, sfGFP (PDB ID: 2B3P) was chosen as a model protein because the protein is a robust folder more resistant to denaturation than many proteins, 51 has a known crystal structure, 52 and has known plasticity at many AA positions that allow reconstruction of the protein surface. 47,53 In this study, sfGFP protein was mutated to create a panel of mutants with differing electrostatic patch sizes on their surfaces.…”