Bioorthogonal Chemistry Enables Single-Molecule FRET Measurements of Catalytically Active Protein Disulfide Isomerase

Mathivanan Chinnaraj, David A. Barrios, Carl Frieden, Tomasz Heyduk, Robert Flaumenhaft, Nicola Pozzi

Research output: Contribution to journalArticlepeer-review

5 Scopus citations

Abstract

Folding of newly synthesized proteins in the endoplasmic reticulum is assisted by several families of enzymes. One such family is the protein disulfide isomerases (PDIs). PDIs are oxidoreductases, capable of forming new disulfide bonds or breaking existing ones. Structural information on PDIs unbound and bound to substrates is highly desirable for developing targeted therapeutics, yet it has been difficult to obtain by using traditional approaches because of their relatively large size and remarkable flexibility. Single-molecule FRET (smFRET) could be a powerful tool to study PDIs’ structure and dynamics under conditions relevant to physiology, but its implementation has been hindered by technical challenges of position-specific fluorophore labeling. We have overcome this limitation by site-specifically engineering fluorescent dyes into human PDI, the founding member of the family. Proof-of-concept smFRET measurements of catalytically active PDI demonstrate, for the first time, the feasibility of this approach, expanding the toolkit for structural studies of PDIs.

Original languageEnglish
Pages (from-to)134-138
Number of pages5
JournalChemBioChem
Volume22
Issue number1
DOIs
StatePublished - Jan 5 2021

Keywords

  • FRET
  • click chemistry
  • oxidoreductases
  • single-molecule studies
  • structural biology

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