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. Published by Cold Spring Harbor Laboratory Press.
|Number of pages||7|
|State||Published - Mar 2006|
Bibliographical noteFunding Information:
Funding. This research was supported by the German Research Foundation (Deutsche Forschungsgemeinschaft, SFB Transregio 130, Project 15).
- Alternating laser excitation
- Fluorescence resonance energy transfer
- Fluorescence-aided molecular sorting
- Protein labeling
- Single-molecule spectroscopy