A Biophysical Study of Clathrin Utilizing Light Scattering, Neutron Scattering and Structure Based Computer Modeling
A Biophysical Study of Clathrin Utilizing Light Scattering, Neutron Scattering and Structure Based Computer Modeling
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Date
2007-04-27
Authors
Ferguson, Matthew Lee
Advisor
Nossal, Ralph J
Losert, Woflgang
Losert, Woflgang
Citation
DRUM DOI
Abstract
A principal component in the protein coats of certain post-golgi and endocytic
vesicles is clathrin, which appears as a three-legged heteropolymer (known as a triske-
lion) that assembles into polyhedral baskets principally made up of pentagonal and
hexagonal faces. In vitro, this assembly depends on the pH, with baskets forming
more readily at low pH and less readily at high pH. We have developed procedures,
based on static and dynamic light scattering, to determine the radius of gyration, Rg,
and hydrodynamic radius, RH, of isolated triskelia under conditions where basket
assembly occurs. Calculations based on rigid molecular bead models of a triskelion
show that the measured values can be accounted for by bending of the legs and a
puckering at the vertex. We also show that the values of Rg and RH measured for
clathrin triskelia in solution are qualitatively consistent with the conformation of
an individual triskelion that is part of a "D6 barrel" basket assembly measured by
cryo-EM tomography.
We extended this study by performing small angle neutron scattering (SANS)
experiments on isolated triskelia in solution under conditions where baskets do not
assemble. SANS experiments were consistent with previous static light scattering ex-
periments but showed a shoulder in the scattering function at intermediate q-values
just beyond the central diffraction peak (the Guinier regime). Theoretical calcula-
tions based on rigid bead models of a triskelion showed well-defined features in this
region different from the experiment. A flexible bead-spring model of a triskelion
and Brownian dynamics simulations were used to generate a time averaged scattering
function. This model adequately described the experimental data for flexibilities close
to previous estimates from the analysis of electron micrographs.