X-ray crystal structures of amaranthin: Ligand-free and in complex with the T antigen disaccharide.
dc.contributor.author | Transue, Thomas Raoul | en_US |
dc.contributor.advisor | Saper, Mark A. | en_US |
dc.date.accessioned | 2014-02-24T16:24:16Z | |
dc.date.available | 2014-02-24T16:24:16Z | |
dc.date.issued | 1995 | en_US |
dc.identifier.other | (UMI)AAI9610253 | en_US |
dc.identifier.uri | http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqm&rft_dat=xri:pqdiss:9610253 | en_US |
dc.identifier.uri | https://hdl.handle.net/2027.42/104874 | |
dc.description.abstract | Amaranthin is a lectin or carbohydrate-binding protein isolated from the seeds of the grain Amaranthus caudatus. Previous biochemical studies showed it to be a homodimer of 66 kDa with highest specificity for the Thompsen-Friedenreich or T-antigen disaccharide (Gal $\beta$1,3 GalNAc $\alpha$-O-R), a surface antigen of carcinoma cells. Each dimer stoichiometrically binds two disaccharides, but unlike many other plant lectins, requires no divalent metal ions for binding and is free of covalent glycosylation. We have grown crystals by micro-vapor diffusion and collected a 92% complete native data set to 2.2 A resolution. The crystals are orthorhombic with space group $P2\sb12\sb12\sb1$ with $a=111.3,\ b=99.1,$ and $c=66.4$ A and contain two monomers per asymmetric unit. Multiple isomorphous replacement phases to 3.0 A were calculated from trimethyllead acetate, platinum (IV) chloride, and potassium uranyl fluoride derivatives. Electron density maps were improved by solvent flattening and density averaging about the non-crystallographic two-fold axis. A 270-residue polyalanine model was built into the resulting density. The complete model, including 5,340 non-hydrogen atoms in the dimer, was refined to an R-factor of 16.9% for 31,047 reflections between 10 and 2.2 A. Each subunit contains two highly homologous domains separated by a short 3$\sb{10}$ helix. Each domain consists of a six-stranded anti-parallel $\beta$-barrel and three two-stranded $\beta$-hairpins. This fold is characteristic of the $\beta$-trefoil structural family which includes fibroblast growth factor, interleukin-1, hisactophilin, Kunitz-type trypsin inhibitors from Erythrina and soybean, and the lectin (B) chain from the cytotoxic heterodimer ricin. Complexes of amaranthin with the tight-binding T-antigen disaccharide ($\alpha$-O-benzyl glycoside, $\rm IC\sb{50}=1.6\ \mu M$) and the weaker-binding $\alpha$-methyl N-acetylgalactosaminide (GalNAc) were solved and refined to R-factors of 19.4% (2.2 A) and 16.0% (2.5 A), respectively. Both dimer subunits contact the terminal galactose of the disaccharide, while only one subunit interacts with the GalNAc of the di- or monosaccharide. Amaranthin forms hydrogen bonds with hydroxyl groups of both ligands, but lacks contact between an aromatic side chain and the hydrophobic face of the sugar seen in other lectin-sugar complexes. | en_US |
dc.format.extent | 134 p. | en_US |
dc.subject | Chemistry, Biochemistry | en_US |
dc.subject | Biophysics, Medical | en_US |
dc.title | X-ray crystal structures of amaranthin: Ligand-free and in complex with the T antigen disaccharide. | en_US |
dc.type | Thesis | en_US |
dc.description.thesisdegreename | PhD | en_US |
dc.description.thesisdegreediscipline | Biological Chemistry | en_US |
dc.description.thesisdegreegrantor | University of Michigan, Horace H. Rackham School of Graduate Studies | en_US |
dc.description.bitstreamurl | http://deepblue.lib.umich.edu/bitstream/2027.42/104874/1/9610253.pdf | |
dc.description.filedescription | Description of 9610253.pdf : Restricted to UM users only. | en_US |
dc.owningcollname | Dissertations and Theses (Ph.D. and Master's) |
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