Bioorthogonal Chemistry Enables Single‐Molecule FRET Measurements of Catalytically Active Protein Disulfide Isomerase
| Autor: | Carl Frieden, Robert Flaumenhaft, Tomasz Heyduk, Nicola Pozzi, Mathivanan Chinnaraj, David A. Barrios |
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| Rok vydání: | 2020 |
| Předmět: |
Fluorophore
Protein Conformation Protein Disulfide-Isomerases Molecular Dynamics Simulation Protein Engineering 010402 general chemistry 01 natural sciences Biochemistry Article chemistry.chemical_compound Fluorescence Resonance Energy Transfer Humans Protein disulfide-isomerase Molecular Biology Fluorescent Dyes 010405 organic chemistry Organic Chemistry Single-molecule FRET 0104 chemical sciences Folding (chemistry) Förster resonance energy transfer chemistry Structural biology Biocatalysis Click chemistry Biophysics Molecular Medicine Bioorthogonal chemistry |
| Zdroj: | Chembiochem |
| ISSN: | 1439-7633 1439-4227 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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