Site-specific incorporation of a (19)F-amino acid into proteins as an NMR probe for characterizing protein structure and reactivity.
19F 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 19F NMR chemical shifts. Subtle protein conformational changes were detected near the active site and at long distances (25 Angstrom). 19F signal sensitivity and resolution was also sufficient to differentiate protein environments in vivo. Since there has been interest in using 19F-labeled proteins in solid-state membrane protein studies, folding studies, and in vivo studies, this general method for genetically incorporating a 19F-label into proteins of any size in Escherichia coli should have broad application beyond that of monitoring protein conformational changes.
Jackson
Jennifer C
JC
Department of Chemistry, Franklin and Marshall College, P.O. Box 3003, Lancaster, Pennsylvania 17604, USA.
Hammill
Jared T
JT
Mehl
Ryan A
RA
D000596:Amino Acids / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray / Q000379:methods; D004926:Escherichia coli / Q000737:chemistry / Q000378:metabolism; D005461:Fluorine / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D010649:Phenylalanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D013194:Staining and Labeling
C557397:4-(trifluoromethyl)phenylalanine; D000596:Amino Acids; D011506:Proteins; D005461:Fluorine; C027218:3-fluorophenylalanine; D010649:Phenylalanine