An often limiting factor for studying protein folding by single-molecule fluorescence resonance energy transfer (FRET) is the ability to site-specifically introduce a photostable organic FRET donor (D) and a complementary acceptor (A) into a polypeptide chain. Using alternating-laser excitation and chymotrypsin inhibitor 2 as a model, we show that chemical labeling of a unique cysteine, followed by enzymatic modification of a reactive glutamine in an N-terminally appended substrate sequence recognition tag for transglutaminase (TGase) affords stoichiometrically D-/A-labeled protein suitable for single-molecule FRET experiments. Thermodynamic data indicate that neither the presence of the TGase tag nor D/A labeling perturbs protein stability. As the N terminus in proteins is typically solvent accessible, a TGase tag can (in principle) be appended to any protein of interest by genetic engineering. Two-step chemical/enzymatic labeling may thus represent a simple, low-cost, and widely available strategy for D/A labeling of proteins for FRET-based single-molecule protein folding studies, even for non-protein-experts laboratories. Study holds ProTherm entries: 20569, 20570, 20571 Extra Details: protein labeling; transglutaminase; fluorescence resonance energy transfer; single-molecule spectroscopy; alternating laser excitation; fluorescence-aided molecular sorting
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:52 p.m.
|Number of data points
|Chymotrypsin inhibitor 2 ; Subtilisin-chymotrypsin inhibitor-2A
|Experimental Assay: m ; Experimental Assay: dG_H2O
|Mutations for sequence SSVEKKPEGVNTGAGDRHNLKTEWPELVGKSVEEAKKVILQDKPEAQIIVLPVGTIVTMEYRIDRVRLFVDKLDNIAEVPRVG