Structural investigations of the cancer-associated laminin binding protein and Nos L : a novel copper binding protein
Taubner, Lara Marie.
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This thesis consists of two distinct projects, one on the metastasis-associated laminin binding protein and the other on the putative copper chaperone NosL, both related by the common aim of investigation of the relationship between protein structure and function using nuclear magnetic resonance techniques. In the first part of this dissertation, the role that the metastasis-associated laminin binding protein or LBP plays in the spread and development of cancer was investigated. Functional domains of LBP were delineated by limited proteolysis, overexpressed, and then assayed for their ability to bind to the previously identified in vivo binding partner laminin. These assays demonstrated that, at least under the conditions used in this assay, binding to laminin was localized to domain 137-230, a region that encompasses a previously identified binding site known as Peptide G. This protein, like the full-length recombinant laminin binding protein, aggregated under conditions used for nuclear magnetic resonance experiments and therefore could not be analyzed with this technique. Contrary to previous findings on a synthetic peptide corresponding to residues 205-229, this sequence within the context of the 200-295 construct demonstrated no laminin binding activity. Furthermore, the peptide lacked the predicted alpha-helical content and tertiary structure as ascertained by nuclear magnetic resonance and by circular dichroism spectroscopy. A potential role for the disorder exhibited by this region of LBP is proposed, and suggests possible new functions for the laminin binding protein in angiogenesis. NosL, the subject of the second part of this thesis, is a highly conserved copper(I) binding lipoprotein encoded by the nitrous oxide reductase (nos) gene cluster of denitrifying bacteria. To identify functional features and structural homologues of this protein, the structure of apo NosL Was solved using nuclear magnetic resonance techniques. The high-resolution structure of NosL consists of one four-strand antiparallel beta sheet, one three strand antiparallel beta sheet and two alpha-helices organized in a twisted butterfly-like fold that is structurally homologous to MerB, an alkyl mercury lyase. Chemical perturbation mapping performed on the copper(I)=protein defined regions of NosL potentially involved in copper binding, and thus allowed preliminary identification of the copper-binding ligand Met 109.