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Title: Three-dimensional structure of cerebellar inositol 1,4,5-trisphosphate receptors
Author: Fernando, Colombapatabendige Pabhashi Lasanthi
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2006
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The objectives of the present study were to determine the location of the IP3-binding sites and IP3-induced conformational changes. As IP3 is too small to be visualized in electron microscopy (EM) and single particle analysis, 2-aminoethyl-IP3 was conjugated to undecagold (sulf-N-hydroxysuccinimido undecagold) to provide a small-diameter (0.8 nm) tag for visualization. IP3R were purified from rat cerebella using affinity chromatography and sucrose density gradient centrifugation methods. The latter confirms that IP3R retain a tetrameric state. Silver staining revealed a single band of 260 kDa, consistent with the expected size for IP3R. Equilibrium competition binding with 3H-IP3 demonstrates that both IP3 and undecagold-IP3 binds with high affinity to purified IP3R. In separate experiments, purified IP3Rs were incubated with undecagold-IP3 and IP3 to obtain two independent data sets (of ~ 4000 images) for difference mapping. After negative staining, micrographs were collected at x35,000 magnification and digitized to a pixel size of 5.44 Å. All subsequent image processing was done using IMAGIC V software. Difference mapping using independent 3D reconstructions of cerebellar IP3R wit IP3 bound (with and without conjugated-undecagold) revealed a central cytoplasmic location for the undecagold. Docking of the modelled undecagold-IP3 and X-ray crystal structure of the IP3-binding cores suggests the IP3-binding pockets to be located ~8 nm from the putative pore, with an inter-subunit spacing of ~5 nm. Comparison of these structures with the previously published unliganded IP3R structure reveals large-scale IP3-induced conformational changes, leading to a speculative scheme for IP3-induced channel gating.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available