Monitoring endocytic trafficking of anthrax lethal factor by precise and quantitative protein labeling.
Coupling the genetic code expansion technique with bioorthogonal reactions enables precise control over the conjugation site as well as the choice of fluorescent probes during protein labeling. However, the advantages of this strategy over bulky and rigid fluorescent proteins (FPs) remain to be fully explored. Here we applied site-specific bioorthogonal labeling on anthrax lethal factor (LF) to visualize its membrane translocation inside live cells. In contrast to the previously reported FP tags that significantly perturbed LF's membrane trafficking, our precisely and quantitatively labeled LF exhibited an endocytic activity comparable to wild-type LF. This allowed time-lapse imaging of LF's natural translocation process from host cell membrane to cytosol, which revealed molecular details of its virulence mechanism. Our strategy is generally applicable for monitoring intracellular protein membrane translocation that is difficult to access using conventional protein labeling methodologies.
Beijing National Laboratory for Molecular Sciences, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education, Synthetic and Functional Biomolecules Center, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871 (China).
Siqi
S
Zheng
Gong
G
Zhang
Jie
J
Li
Peng R
PR
Chen
D000595:Amino Acid Sequence; D000942:Antigens, Bacterial / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D001427:Bacterial Toxins / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis; D005456:Fluorescent Dyes; D014644:Genetic Variation; D008954:Models, Biological; D008969:Molecular Sequence Data; D021381:Protein Transport
D000942:Antigens, Bacterial; D001427:Bacterial Toxins; D005456:Fluorescent Dyes; C030325:anthrax toxin
anthrax lethal factor; bioorthogonal reactions; genetic code expansion; membrane translocation; protein labeling