Site-Specific Incorporation of a 19 F-Amino Acid into Proteins as an NMR Probe for Characterizing Protein Structure and Reactivity Jennifer C. Jackson, Jared T. Hammill, and Ryan A. Mehl* Contribution from the Department of Chemistry, Franklin and Marshall College, P.O. Box 3003, Lancaster, PennsylVania 17604 Received June 30, 2006; E-mail: rmehl@fandm.edu Abstract: 19 F NMR is a powerful tool for monitoring protein conformational changes and interactions; however, the inability to site-specifically introduce fluorine labels into proteins of biological interest severely limits its applicability. Using methods for genetically directing incorporation of unnatural amino acids, we have inserted trifluoromethyl-L-phenylalanine (tfm-Phe) into proteins in vivo at TAG nonsense codons with high translational efficiency and fidelity. The binding of substrates, inhibitors, and cofactors, as well as reactions in enzymes, were studied by selective introduction of tfm-Phe and subsequent monitoring of the 19 F NMR chemical shifts. Subtle protein conformational changes were detected near the active site and at long distances (25 Å). 19 F signal sensitivity and resolution was also sufficient to differentiate protein environments in vivo. Since there has been interest in using 19 F-labeled proteins in solid-state membrane protein studies, folding studies, and in vivo studies, this general method for genetically incorporating a 19 F-label into proteins of any size in Escherichia coli should have broad application beyond that of monitoring protein conformational changes. Introduction The ability to monitor inhibitor binding events in protein structure/activity relationship studies as well as conformational changes and dynamics of proteins that are too large for conventional protein NMR would advance biochemical research. 1-3 The high sensitivity of 19 F to surrounding environments, 100% natural abundance, and high sensitivity to NMR detection (83% that of 1 H) has made 19 F NMR spectroscopy useful for investigating protein structure and dynamics. 1,4-7 The simplicity of observing hypersensitive 19 F chemical shifts by NMR makes it an exquisite tool for monitoring protein movements resulting from small-molecule binding, covalent modification, or protein interactions. 8-11 The ability to uniformly label any single site in a protein in vivo with a 19 F-amino acid will enable the study of large proteins with chemical clarity. Site-specific in vivo incorporation of fluorine into proteins at high yield will advance protein structural work, 3 solid-state studies, 12 and inhibitor design. 13,14 Current methods to incorporate 19 F-labeled amino acids into proteins suffer from certain limitations. For example, semisyn- thetic incorporation enables high fidelity at specific sites but becomes impractical when medium to large proteins are needed. The use of natural translational machinery to force fluorinated mimics of Tyr, Trp, Phe, Met, and Leu into their natural codons can produce large, 19 F-labeled proteins. However, this method of incorporation alters all locations of one amino acid simul- taneously, resulting in structural perturbation and overlapping of 19 F signals in large proteins. 1,5,15,16 Incorporation of fluori- nated mimics rarely approaches 95% at one site when relying on natural machinery. 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