A Biophysical Study of Clathrin Utilizing Light Scattering, Neutron Scattering and Structure Based Computer Modeling

dc.contributor.advisorNossal, Ralph Jen_US
dc.contributor.advisorLosert, Woflgangen_US
dc.contributor.authorFerguson, Matthew Leeen_US
dc.contributor.publisherDigital Repository at the University of Marylanden_US
dc.contributor.publisherUniversity of Maryland (College Park, Md.)en_US
dc.description.abstractA 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.en_US
dc.format.extent2434672 bytes
dc.subject.pqcontrolledPhysics, Molecularen_US
dc.subject.pqcontrolledBiophysics, Generalen_US
dc.subject.pqcontrolledPhysics, Molecularen_US
dc.subject.pquncontrolledlight scatteringen_US
dc.subject.pquncontrolledsmall angle neutron scatteringen_US
dc.subject.pquncontrolledprotein structureen_US
dc.subject.pquncontrolledprotein flexibilityen_US
dc.subject.pquncontrolledreceptor mediated endocytosisen_US
dc.titleA Biophysical Study of Clathrin Utilizing Light Scattering, Neutron Scattering and Structure Based Computer Modelingen_US


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