College Park, Maryland June 6 - 10 , 2004
WP3: Using Deuterated Actin to Reveal the Conformation of Ca2+-Activated Intact Gelsolin Bound to Monomeric and Filamentous Actin by Small Angle Neutron Scattering.
Ashish, J.K. Krueger (UNC Charlotte Chemistry Department)
This work attempts to elucidate the conformational changes of Ca2+-activated gelsolin when it binds to actin through the use of small angle neutron scattering contrast variation experiments using specifically deuterated actin. In the presence of Ca2+, gelsolin binds to monomeric G-actin in a 1:2 molar ratio, effectively catalyzing the nucleation step in actin polymerization. In an apparently contradictory role Ca2+-gelsolin also participates in severing the filamentous F-actin and capping the resultant fragments. Gelsolin is comprised of six structurally similar domains, G1 through G6. In the Ca2+-free inactive state, these domains of intact gelsolin are packed in a manner where none of the actin-binding sites are accessible. Activation by Ca2+ causes large conformational changes altering the relative positions and orientations of the six domains, which exposes several actin-binding surfaces. We have small-angle x-ray scattering data that follows the conformational changes within gelsolin as a result of binding Ca2+ and although new x-ray crystallographic data has been obtained on the N- and C-terminal halves of gelsolin +/- an actin monomer, there is no direct structural information on intact Ca2+-activated gelsolin or intact Ca2+-activated gelsolin bound to either two monomers of G-actin or to filamentous F-actin. For our neutron scattering studies on the gelsolin-actin complexes, deuterated actin was obtained from the slime mold Dictyostelium discoideum, which was fed deuterated Escherichia coli. The actin was purified by ion-exchange and gel filtration chromatography. NMR and mass spectroscopy on non-deuterated and deuterated actin was used to determine the level of actin deuteration. Using small-angle X-ray scattering, we will first optimize the experimental conditions to obtain structural data of Ca2+-activated gelsolin bound to two actin monomers. Small-angle neutron scattering experiments are planned in which we will vary solvent's deuterium composition and use the resultant data to extract out the basic scattering function for the actin-bound Ca2+-gelsolin. Comparison of the molecular shapes obtained from the neutron scattering data with those obtained from X-ray studies, will provide insight into the mechanism by which Ca2+-gelsolin interacts with, and regulates actin polymerization.
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