TY - JOUR
T1 - Unraveling protein-protein interactions in clathrin assemblies via atomic force spectroscopy
AU - Jin, Albert J.
AU - Lafer, Eileen M.
AU - Peng, Jennifer Q.
AU - Smith, Paul D.
AU - Nossal, Ralph
N1 - Funding Information:
We thank Dr. Svetlana Kotova (National Institutes of Health (NIH), currently Laboratory of Modified Polymer Systems, N.N.Semenov Institute of Chemical Physics of the Russian Academy of Sciences, Moscow, Russia) and Dr. Kondury Prasad (University of Texas Health Science Center at San Antonio) for contributions during the early stage of this study and technical assistance. We also are very grateful to Dr. Dan Sackett (NIH) for helpful comments and technical assistance. This work was supported in part by an extramural grant (EML, NIH-NINDS NS029051) and by the intramural research programs of the National Institute of Biomedical Imaging and Bioengineering (NIBIB) and the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD).
PY - 2013/3/1
Y1 - 2013/3/1
N2 - 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.
AB - 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.
KW - Atomic force microscopy (AFM)
KW - Clathrin triskelion and clathrin-coated vesicles
KW - Macromolecular assembly
KW - Protein interaction and protein folding
KW - Single molecular force spectroscopy (SMFS)
KW - Single particle force spectroscopy (SPFS)
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U2 - 10.1016/j.ymeth.2012.12.006
DO - 10.1016/j.ymeth.2012.12.006
M3 - Article
C2 - 23270814
AN - SCOPUS:84884484357
VL - 59
SP - 316
EP - 327
JO - Methods
JF - Methods
SN - 1046-2023
IS - 3
ER -