Probing Local Environments with the Infrared Probe: l-4-Nitrophenylalanine

Abstract

The genetic incorporation of unnatural amino acids (UAAs) with high efficiency and fidelity is a powerful tool for the study of protein structure and dynamics with site-specificity in a relatively nonintrusive manner. Here, we illustrate the ability of L-4-nitrophenylalanine to serve as a sensitive IR probe of local protein environments in the 247 residue superfolder green fluorescent protein (sfGFP). Specifically, the nitro symmetric stretching frequency of L-4-nitrophenylalanine was shown to be sensitive to both solvents that mimic different protein environments and (15)N isotopic labeling of the three-atom nitro group of this UAA. (14)NO(2) and (15)NO(2) variants of this UAA were incorporated utilizing an engineered orthogonal aminoacyl-tRNA synthetase/tRNA pair into a solvent exposed and a partially buried position in sfGFP with high efficiency and fidelity. The combination of isotopic labeling and difference FTIR spectroscopy permitted the nitro symmetric stretching frequency of L-4-nitrophenylalanine to be experimentally measured at either site in sfGFP. The (14)NO(2) symmetric stretching frequency red-shifted 7.7 cm(-1) between the solvent exposed and partially buried position, thus illustrating the ability of this UAA to serve as an effective IR probe of local protein environments.

Sensitive, Site-Specific, and Stable Vibrational Probe of Local Protein Environments: 4-Azidomethyl-l-Phenylalanine

“…The codon-optimized gene containing a C-terminal 6-His affinity tag for wild-type sfGFP (wt-sfGFP) 18,23,24 was inserted into pBadA generating pBad-sfGFP . The codons for Y75 and D134 were individually replaced by site-directed mutagenesis with the amber stop codon (TAG) generating pBad-sfGFP-75TAG and pBad-sfGFP-134TAG , respectively.…”

“…The cells from the autoinduction media were collected by centrifugation after shaking at 37°C for 24 – 30 hrs and the expressed protein was purified using TALON cobalt ion-exchange chromatography (Clontech) similar to previous procedures. 5,23,24 …”

Playing with the Molecules of Life

“…The incorporation of deuterium, 170 cyano, 171 nitro, 172 and azido 59 IR active probes into proteins introduces unique vibrational modes that do not overlap those present in the canonical amino acids. These localized probes have facilitated investigations of enzymatic catalysis, 170 solvent exposure 173, 174 and receptor activation using IR spectroscopy.…”

Site-Specific Spectroscopic Reporters of the Local Electric Field, Hydration, Structure, and Dynamics of Biomolecules

“…Unfortunately, the entire body of deserving studies cannot be reviewed in this section and the interested reader may find other very interesting applications of these probes in the references cited. 97-113 …”