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.

Membrane Fluctuations Destabilize Clathrin Protein Lattice Order

“…This value exceeds a predicted minimum binding strength for stabilizing vesicles in vivo (27) and is half as strong as an estimate based on fitting a thermodynamic model to cage-size distributions in vitro (28). It is also close to an order-of-magnitude estimate of 10 k B T based on atomic force measurements of triskelion removal from assembled structures (29). Given the approximations inherent in each of these experimental fits and the depen-dence of the affinity on environmental conditions, our chosen value of 6.5 k B T is within a realistic range.…”

Membrane Fluctuations Destabilize Clathrin Protein Lattice Order

“…This value exceeds a predicted minimum binding strength for stabilizing vesicles in vivo (27) and is half as strong as an estimate based on fitting a thermodynamic model to cage-size distributions in vitro (28). It is also close to an order-of-magnitude estimate of 10 k B T based on atomic force measurements of triskelion removal from assembled structures (29). Given the approximations inherent in each of these experimental fits and the dependence of the affinity on environmental conditions, our chosen value of 6.5 k B T is within a realistic range.…”

Atomic force microscopy of food assembly: Structural and mechanical insights at the nanoscale and potential opportunities from other fields

“…In general, the energy required for parting the proteins is less than that of the protein's unfolding energy and ideal constant-velocity experiments yield a single adhesion (or unbinding) peak (Liu et al, 2016). If unfolding occurs prior to unbinding, only the last or "jumpoff" peak is considered (Burgain et al, 2014b;Jin, Lafer, Peng, Smith, & Nossal, 2013). Among food proteins, the interaction between two cruciferin or napin plant proteins was measured this way (Fahs & Louarn, 2013).…”