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

Title of dissertation: A BIOPHYSICAL STUDY OF CLATHRIN UTILIZING LIGHT SCATTERING, NEUTRON SCATTERING AND STRUCTURE BASED COMPUTER MODELING Matthew Lee Ferguson, Doctor of Philosophy, 2007 Dissertation directed by: Dr. Ralph Nossal National Institute of Child Health and Human Development and Professor Wolfgang Losert Department of Physics 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 triskelion) 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 experiments but showed a shoulder in the scattering function at intermediate q-values just beyond the central diffraction peak (the Guinier regime). Theoretical calculations 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. A BIOPHYSICAL STUDY OF CLATHRIN UTILIZING LIGHT SCATTERING, NEUTRON SCATTERING AND STRUCTURE BASED COMPUTER MODELING by Matthew Lee Ferguson Dissertation submitted to the Faculty of the Graduate School of the University of Maryland, College Park in partial fulfillment of the requirements for the degree of Doctor of Philosophy 2007 Advisory Committee: Dr. Ralph J. Nossal, Co-Chair/Advisor Professor Wolfgang Losert, Co-Chair/Advisor Dr. Dan L. Sackett Professor Sandra Greer Professor J. Robert Dorfman Professor Robert W. Gammon c © Copyright by Matthew Lee Ferguson 2007