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Site-specific labeling of proteins for single-molecule FRET by combining chemical and enzymatic modification.
- Source :
- Protein Science: A Publication of the Protein Society; 2006, Vol. 15 Issue 3, p640-646, 7p
- Publication Year :
- 2006
-
Abstract
- 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. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 09618368
- Volume :
- 15
- Issue :
- 3
- Database :
- Complementary Index
- Journal :
- Protein Science: A Publication of the Protein Society
- Publication Type :
- Academic Journal
- Accession number :
- 90754962
- Full Text :
- https://doi.org/10.1110/ps.051851506