Dual Unnatural Amino Acid Incorporation and Click-Chemistry Labeling to Enable Single-Molecule FRET Studies of p97 Folding.
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.
The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037, USA.
Taehyung C
TC
Lee
College of Pharmacy, Department of Pharmacology and Toxicology, The University of Arizona, Tucson, AZ, 85721, USA.
Minjin
M
Kang
The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037, USA.
Chan Hyuk
CH
Kim
The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037, USA.
Peter G
PG
Schultz
College of Pharmacy, Department of Pharmacology and Toxicology, The University of Arizona, Tucson, AZ, 85721, USA. chapman@pharmacy.arizona.edu.
Eli
E
Chapman
The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA, 92037, USA. deniz@scripps.edu.
Ashok A
AA
Deniz
D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D057930:Click Chemistry; D004926:Escherichia coli / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D006801:Humans; D006834:Hydrazines / Q000737:chemistry; D017510:Protein Folding; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D000074405:Valosin Containing Protein
C417561:Alexa 488 hydrazide; D000596:Amino Acids; D018797:Cell Cycle Proteins; D006834:Hydrazines; D011994:Recombinant Proteins; D000251:Adenosine Triphosphatases; D000074405:Valosin Containing Protein
FRET; click chemistry; p97; protein folding; unnatural amino acid