Dual Unnatural Amino Acid Incorporation and Click‐Chemistry Labeling to Enable Single‐Molecule FRET Studies of p97 Folding. (26th April 2016)

• Record Type: Journal Article
• Authors: Lee, Taehyung C.; Kang, Minjin; Kim, Chan Hyuk; Schultz, Peter G.; Chapman, Eli; Deniz, Ashok A.
• Is Part Of: Chembiochem. Volume 17:Number 11(2016)
• Page Start: 981
• Page End: 984
• Publication Date: 2016-04-26
• Subjects: click chemistry -- FRET -- p97 -- protein folding -- unnatural amino acid; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pharmaceutical chemistry -- Periodicals; 572
• DOI: 10.1002/cbic.201500695
• Languages: English
• ISSNs: 1439-4227

Abstract

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. Abstract : 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.