Atomic force microscopy (AFM) and single molecule force spectroscopy  (SMFS) were used to characterize intermolecular interactions and domain  structures of clathrin triskelions, principal structural units of  clathrin-coated vesicles (CCVs)  involved in receptor-mediated endocytosis. We employed AFM imaging to  quantify the number of triskelion dimers and higher-order oligomers that  arise when buffer pH is lowered to favor triskelion-triskelion  interactions. We used AFM-SMFS to study purified bovine-brain  CCVs, reassembled clathrin cages, and individual triskelions. For  triskelions, we detected internal energy barriers against the unfolding  of individual alpha-helix hairpins containing ca. 30 amino acid  residues, and discerned the cooperative unraveling of  up to five hairpin domains. We performed large-scale data analytics to  determine dynamic domain disruption forces ranging from 10s of pN to  about 500 pN. This force increases continuously as the stretching rate  increases, in accordance with the Bell model.

Assessing clathrin-clathrin interactions and clathrin hair-pin folds by atomic force spectroscopy

244th National Meeting (2012)

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