Site-specific labeling of proteins for single-molecule FRET by combining chemical and enzymatic modification

Marcus Jäger, Eyal Nir, Shimon Weiss

Research output: Contribution to journalArticlepeer-review

50 Scopus citations

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. Published by Cold Spring Harbor Laboratory Press.

Original languageEnglish
Pages (from-to)640-646
Number of pages7
JournalProtein Science
Volume15
Issue number3
DOIs
StatePublished - 1 Mar 2006
Externally publishedYes

Keywords

  • Alternating laser excitation
  • Fluorescence resonance energy transfer
  • Fluorescence-aided molecular sorting
  • Protein labeling
  • Single-molecule spectroscopy
  • Transglutaminase

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