Unraveling protein-protein interactions in clathrin assemblies via atomic force spectroscopy

Albert J. Jin, Eileen M. Lafer, Jennifer Q. Peng, Paul D. Smith, Ralph Nossal

Research output: Contribution to journalArticle

4 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)316-327
Number of pages12
JournalMethods
Volume59
Issue number3
DOIs
StatePublished - Mar 1 2013

Keywords

  • Atomic force microscopy (AFM)
  • Clathrin triskelion and clathrin-coated vesicles
  • Macromolecular assembly
  • Protein interaction and protein folding
  • Single molecular force spectroscopy (SMFS)
  • Single particle force spectroscopy (SPFS)

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)

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