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

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

Published In Chembiochem, (2016 06 02)

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.

PubMed ID: 27115850 Exiting the NIEHS site

MeSH Terms: Adenosine Triphosphatases/chemistry; Adenosine Triphosphatases/genetics; Adenosine Triphosphatases/metabolism*; Amino Acids/chemistry; Amino Acids/metabolism*; Cell Cycle Proteins/chemistry; Cell Cycle Proteins/genetics; Cell Cycle Proteins/metabolism*; Click Chemistry; Escherichia coli/metabolism; Fluorescence Resonance Energy Transfer; Humans; Hydrazines/chemistry; Protein Folding; Recombinant Proteins/biosynthesis; Recombinant Proteins/chemistry; Recombinant Proteins/isolation & purification; Valosin Containing Protein

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