Dual Unnatural Amino Acid Incorporation and Click‐Chemistry Labeling to Enable Single‐Molecule FRET Studies of p97 Folding |
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Authors: | Taehyung C. Lee Dr. Minjin Kang Dr. Chan Hyuk Kim Prof. Dr. Peter G. Schultz Prof. Dr. Eli Chapman Ashok A. Deniz |
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Affiliation: | 1. The Scripps Research Institute, La Jolla, CA, USA;2. College of Pharmacy, Department of Pharmacology and Toxicology, The University of Arizona, Tucson, AZ, USA |
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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. |
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Keywords: | click chemistry FRET p97 protein folding unnatural amino acid |
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