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

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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 intra-domain 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.
Publisher
John Wiley & Sons Ltd.
Issue Date
2016-06
Language
English
Article Type
Article
Citation

ChemBioChem, v.17, no.11, pp.981 - 984

ISSN
1439-4227
DOI
10.1002/cbic.201500695
URI
http://hdl.handle.net/10203/281748
Appears in Collection
BS-Journal Papers(저널논문)
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