The genetic incorporation of unnatural amino acids(UAAs) with high efficiency and fidelity is a powerful tool for the study ofprotein structure and dynamics with site-specificity in a relativelynon-intrusive manner. Here, we illustrate the ability of L-4-nitrophenylalanineto serve as a sensitive IR probe of local protein environments in the 247residue superfolder green fluorescent protein (sfGFP). Specifically, the nitrosymmetric stretching frequency of L-4-nitrophenylalanine was shown to besensitive to both solvents that mimic different protein environments and 15Nisotopic labeling of the three-atom nitro group of this UAA.  14NO2 and 15NO2variants of this UAA were incorporated utilizing an engineered aminoacyl-tRNAsynthetase/tRNA pair into a solvent exposed and a partially buried position insfGFP with high efficiency and fidelity. The combination of isotopic labeling and difference FTIR spectroscopypermitted the nitro symmetric stretching frequency of L-4-nitrophenylalanine tobe experimentally measured at either site in sfGFP.  Additional spectroscopic results exploring the utility of otherisotopically labeled UAAs to probe local protein environments withsite-specificity will also be presented.

Probing local protein environments with the infrared probe: L-4-Nitrophenylalanine

241st National Meeting (2011)

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