title: Unraveling protein-protein interactions in clathrin assemblies via atomic force spectroscopy.
abstract: Atomic force microscopy (AFM), single molecule force spectroscopy (SMFS), and single particle force spectroscopy (SPFS) are used to characterize intermolecular interactions and domain structures of clathrin triskelia and clathrin-coated vesicles (CCVs). The latter are involved in receptor-mediated endocytosis (RME) and other trafficking pathways. Here, we subject individual triskelia, bovine-brain CCVs, and reconstituted clathrin-AP180 coats to AFM-SMFS and AFM-SPFS pulling experiments and apply novel analytics to extract force-extension relations from very large data sets. The spectroscopic fingerprints of these samples differ markedly, providing important new information about the mechanism of CCV uncoating. For individual triskelia, SMFS reveals a series of events associated with heavy chain alpha-helix hairpin unfolding, as well as cooperative unraveling of several hairpin domains. SPFS of clathrin assemblies exposes weaker clathrin-clathrin interactions that are indicative of inter-leg association essential for RME and intracellular trafficking. Clathrin-AP180 coats are energetically easier to unravel than the coats of CCVs, with a non-trivial dependence on force-loading rate.
authors.affiliation: Laboratory of Cellular Imaging and Macromolecular Biophysics, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, DHHS, Bethesda, MD 20892, United States. jina@mail.nih.gov
authors.forename: Albert J
authors.initials: AJ
authors.lastname: Jin
authors.forename: Eileen M
authors.initials: EM
authors.lastname: Lafer
authors.forename: Jennifer Q
authors.initials: JQ
authors.lastname: Peng
authors.forename: Paul D
authors.initials: PD
authors.lastname: Smith
authors.forename: Ralph
authors.initials: R
authors.lastname: Nossal
mesh_terms: D000818:Animals; D002417:Cattle; D002966:Clathrin / Q000737:chemistry*; D022163:Clathrin-Coated Vesicles / Q000737:chemistry*; D018625:Microscopy, Atomic Force / Q000379:methods*; D017510:Protein Folding; D025941:Protein Interaction Mapping / Q000379:methods*; D017434:Protein Structure, Tertiary; D013057:Spectrum Analysis / Q000379:methods
chemical_list: D002966:Clathrin