title: 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.
authors.affiliation: Department of Chemistry, Franklin & Marshall College, Lancaster, Pennsylvania 17604-3003, USA.
authors.forename: Emily E
authors.initials: EE
authors.lastname: Smith
authors.forename: Barton Y
authors.initials: BY
authors.lastname: Linderman
authors.forename: Austin C
authors.initials: AC
authors.lastname: Luskin
authors.forename: Scott H
authors.initials: SH
authors.lastname: Brewer
mesh_terms: D049452:Green Fluorescent Proteins / Q000737:chemistry; D007259:Infrared Rays; D007554:Isotopes; D008958:Models, Molecular; D015335:Molecular Probes / Q000737:chemistry*; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry; D017433:Protein Structure, Secondary; D012997:Solvents / Q000737:chemistry
chemical_list: D007554:Isotopes; D015335:Molecular Probes; D012997:Solvents; D049452:Green Fluorescent Proteins; C040239:4-nitrophenylalanine; D010649:Phenylalanine