title: Labeling proteins with fluorophore/thioamide Förster resonant energy transfer pairs by combining unnatural amino acid mutagenesis and native chemical ligation.
abstract: We have recently shown that p-cyanophenylalanine (Cnf) and a thioamide can be used as a minimally perturbing Förster resonant energy transfer (FRET) pair to monitor protein conformation. We have also shown that thioamide analogues of natural amino acids can be incorporated into full-sized proteins through native chemical ligation. For intermolecular studies with Cnf/thioamide FRET pairs, Cnf can be incorporated into proteins expressed in Escherichia coli through unnatural amino acid mutagenesis using a Cnf-specific tRNA synthetase. For intramolecular studies, a Cnf-labeled protein fragment can be expressed in E. coli and then ligated to a thioamide-labeled peptide synthesized on solid phase. This combination of methods allows for rapid access to double-labeled proteins with a minimum of unnecessary chemical synthesis. We demonstrate the utility of this approach by studying the binding of peptides to the protein calmodulin and by determining the orientation of the N- and C-termini in the amyloidogenic protein α-synuclein.
authors.affiliation: Department of Chemistry, University of Pennsylvania, 231 South 34th Street, Philadelphia, Pennsylvania 19104-6323, USA.
authors.forename: Rebecca F
authors.initials: RF
authors.lastname: Wissner
authors.forename: Solongo
authors.initials: S
authors.lastname: Batjargal
authors.forename: Colin M
authors.initials: CM
authors.lastname: Fadzen
authors.forename: E James
authors.initials: EJ
authors.lastname: Petersson
mesh_terms: D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry*; D000604:Amino Acyl-tRNA Synthetases / Q000378:metabolism; D000626:Aminopeptidases / Q000737:chemistry / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004735:Energy Transfer; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015951:Factor Xa / Q000737:chemistry / Q000235:genetics; D005456:Fluorescent Dyes / Q000737:chemistry*; D006801:Humans; D008958:Models, Molecular; D008968:Molecular Conformation; D016296:Mutagenesis; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding; D011492:Protein Hydrolysates / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013854:Thioamides / Q000737:chemistry*; D014357:Trypsin / Q000737:chemistry; D051844:alpha-Synuclein / Q000737:chemistry
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D002147:Calmodulin; D005456:Fluorescent Dyes; D011492:Protein Hydrolysates; D011506:Proteins; D013854:Thioamides; D051844:alpha-Synuclein; D000626:Aminopeptidases; D014357:Trypsin; D015951:Factor Xa; D000604:Amino Acyl-tRNA Synthetases