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Labeling Proteins with Fluorophore/Thioamide Förster Resonant Energy Transfer Pairs by Combining Unnatural Amino Acid Mutagenesis and Native Chemical Ligation

2013; American Chemical Society; Volume: 135; Issue: 17 Linguagem: Inglês

10.1021/ja4005943

1943-2984

Rebecca F. Wissner, Solongo Batjargal, Colin M. Fadzen, E. James Petersson,

RNA and protein synthesis mechanisms

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.