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Clathrin Triskelia Show Evidence of Molecular Flexibility

Matthew L. Ferguson ^* , Kondury Prasad , Hacene Boukari , Dan L. Sackett , Susan Krueger , Eileen M. Lafer  and Ralph Nossal 

^* Department of Physics, University of Maryland, College Park, Maryland; Laboratory of Integrative and Medical Biophysics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland; Department of Biochemistry, University of Texas Health Science Center, San Antonio, Texas; and NIST Center for Neutron Research, National Institute of Standards and Technology, Gaithersburg, Maryland

Correspondence: Address reprint requests to Ralph Nossal, Tel.: 301-435-9233; E-mail: nossalr{at}mail.nih.gov.

The clathrin triskelion, which is a three-legged pinwheel-shaped heteropolymer, is a major component in the protein coats of certain post-Golgi and endocytic vesicles. At low pH, or at physiological pH in the presence of assembly proteins, triskelia will self-assemble to form a closed clathrin cage, or "basket". Recent static light scattering and dynamic light scattering studies of triskelia in solution showed that an individual triskelion has an intrinsic pucker similar to, but differing from, that inferred from a high resolution cryoEM structure of a triskelion in a clathrin basket. We extend the earlier solution studies by performing small-angle neutron scattering (SANS) experiments on isolated triskelia, allowing us to examine a higher q range than that probed by static light scattering. Results of the SANS measurements are consistent with the light scattering measurements, but show a shoulder in the scattering function at intermediate q values (0.016 Å^–1), just beyond the Guinier regime. This feature can be accounted for by Brownian dynamics simulations based on flexible bead-spring models of a triskelion, which generate time-averaged scattering functions. Calculated scattering profiles are in good agreement with the experimental SANS profiles when the persistence length of the assumed semiflexible triskelion is close to that previously estimated from the analysis of electron micrographs.