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

摘要

Many cellular functions are critically dependent on the folding of complex multimeric proteins, such as p97, a hexameric multi-domain AAA+ chaperone. Given p97’s complex architecture, single-molecule Förster Resonance Energy Transfer (smFRET) 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 incorporate 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 demonstrates the strengths of the labeling method. Our results highlight a powerful tool for structural studies of p97 and other large protein machines.