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UBC Theses and Dissertations

Characterization and crystalization of Gelsolin Wang, Hui

Abstract

Actin, a protein existing in all eukaryotic cells, is involved in such cellular processes as cell movement, cell division, apoptosis and so on. Gelsolin, which consists of six structurally similar domains (G1-G6), can regulate these processes by calcium activated disassembly of actin filaments. In inactive gelsolin, the six domains pack into a compact globule so that none of the actin binding sites are accessible. From inactive gelsolin to activated gelsolin, three latches are unlocked to expose actin binding sites. Although the structure of fully activated, intact gelsolin is not available, those of the C-terminal and N-terminal halves of gelsolin, respectively, each bound to one actin molecule, have been solved. These structures may be added on to a model for F-actin to generate a model for a gelsolin-capped actin filament. In order to test these models, we set about to crystallize complexes in which one, two and three actin units bind to one activated intact gelsolin, i.e. GA, GA₂ and GA₃, respectively. This thesis centers on purification, characterization and crystallization of GA, GA₂ and GA₃ complexes. We have crystallized GA, GA₂ and GA₃, respectively, as confirmed by size exclusion chromatography and gel electrophoresis. We also confirmed our GA₂ and GA₃ complexes by MALDI mass spectrometry experiments. Although only the N-terminal half of gelsolin bound to one actin is visible in our GA₂ and GA₃ crystals by X-ray diffraction analysis, the crystals possess a unit cell that is sufficiently large to accommodate the second half of gelsolin bound to a second actin in a variety of different positions. We are still awaiting diffraction data of GA crystals and EDC cross-linked GA₃ crystals. We also soaked GA₂ and GA₃ crystals in Tb³⁺ -containing solutions in order to investigate the exchangeability of Ca²⁺ in these complexes.

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