ArticleProtein science : a publication of the Protein Society2006
Site-specific labeling of proteins for single-molecule FRET by combining chemical and enzymatic modification.
Article in Protein science : a publication of the Protein Society, 2006. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 17 papers.
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Who cites it
17 citing papers in PubMed, 55 citations in OpenAlex.
- Monitoring Single DNA Docking Site Activity with Sequential Modes of an Optoplasmonic Whispering-Gallery Mode Biosensor.Sensors (Basel, Switzerland) · 2025Article
- Integrating single-molecule FRET and biomolecular simulations to study diverse interactions between nucleic acids and proteins.Essays in biochemistry · 2021Review
- FRET-based dynamic structural biology: Challenges, perspectives and an appeal for open-science practices.eLife · 2021Review
- Single-Molecule Analysis of Reverse Transcriptase Enzymes.Cold Spring Harbor perspectives in biology · 2019Review
- LinkingeLife · 2018Article
- Engineering a Prototypic P-type ATPase Listeria monocytogenes Ca(2+)-ATPase 1 for Single-Molecule FRET Studies.Bioconjugate chemistry · 2016Article
- Fluorescent Reporters and Biosensors for Probing the Dynamic Behavior of Protein Kinases.Proteomes · 2015Review
- Single molecule FRET reveals pore size and opening mechanism of a mechano-sensitive ion channel.eLife · 2014Article
- Non-Coding RNAs: Functional Aspects and Diagnostic Utility in Oncology.International journal of molecular sciences · 2013Article
- Catalyst-free and site-specific one-pot dual-labeling of a protein directed by two genetically incorporated noncanonical amino acids.Chembiochem : a European journal of chemical biology · 2012Article
- Dimer-monomer equilibrium of human thymidylate synthase monitored by fluorescence resonance energy transfer.Protein science : a publication of the Protein Society · 2010Article
- Leukotriene BLT2 receptor monomers activate the G(i2) GTP-binding protein more efficiently than dimers.The Journal of biological chemistry · 2010Article
- A general and efficient method for the site-specific dual-labeling of proteins for single molecule fluorescence resonance energy transfer.Journal of the American Chemical Society · 2008Article
- Zinc porphyrin: a fluorescent acceptor in studies of Zn-cytochrome c unfolding by fluorescence resonance energy transfer.Proceedings of the National Academy of Sciences of the United States of America · 2008Article
- Nonequilibrium single molecule protein folding in a coaxial mixer.Biophysical journal · 2008Article
- A practical guide to single-molecule FRET.Nature methods · 2008Review
- Efficient site-specific labeling of proteins via cysteines.Bioconjugate chemistry · 2008Article
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
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.
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