Dual Unnatural Amino Acid Incorporation and Click-Chemistry Labeling to Enable Single-Molecule FRET Studies of p97 Folding

Taehyung C. Lee, Minjin Kang, Chan Hyuk Kim, Peter G. Schultz, Eli Chapman, Ashok A. Deniz

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

Many cellular functions are critically dependent on the folding of complex multimeric proteins, such as p97, a hexameric multidomain AAA+ chaperone. Given the complex architecture of p97, single-molecule (sm) FRET would be a powerful tool for studying folding while avoiding ensemble averaging. However, dual site-specific labeling of such a large protein for smFRET is a significant challenge. Here, we address this issue by using bioorthogonal azide-alkyne chemistry to attach an smFRET dye pair to site-specifically incorporated unnatural amino acids, allowing us to generate p97 variants reporting on inter- or intradomain structural features. An initial proof-of-principle set of smFRET results demonstrated the strengths of this labeling method. Our results highlight this as a powerful tool for structural studies of p97 and other large protein machines. Watching one p97 at a time: The single-molecule biophysical chemistry of p97, a complex, cysteine-rich, essential cellular protein machine, can now be studied using smFRET, thanks to the use of advanced site-specific dye labeling by an unnatural amino acid and click chemistry.

Original languageEnglish (US)
Pages (from-to)981-984
Number of pages4
JournalChemBioChem
Volume17
Issue number11
DOIs
StatePublished - Jun 2 2016

Fingerprint

Click Chemistry
Labeling
Amino Acids
Molecules
Proteins
Coloring Agents
Alkynes
Azides
Cysteine

Keywords

  • click chemistry
  • FRET
  • p97
  • protein folding
  • unnatural amino acid

ASJC Scopus subject areas

  • Biochemistry
  • Organic Chemistry
  • Molecular Medicine
  • Molecular Biology

Cite this

Dual Unnatural Amino Acid Incorporation and Click-Chemistry Labeling to Enable Single-Molecule FRET Studies of p97 Folding. / Lee, Taehyung C.; Kang, Minjin; Kim, Chan Hyuk; Schultz, Peter G.; Chapman, Eli; Deniz, Ashok A.

In: ChemBioChem, Vol. 17, No. 11, 02.06.2016, p. 981-984.

Research output: Contribution to journalArticle

Lee, Taehyung C. ; Kang, Minjin ; Kim, Chan Hyuk ; Schultz, Peter G. ; Chapman, Eli ; Deniz, Ashok A. / Dual Unnatural Amino Acid Incorporation and Click-Chemistry Labeling to Enable Single-Molecule FRET Studies of p97 Folding. In: ChemBioChem. 2016 ; Vol. 17, No. 11. pp. 981-984.
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