Protein-carbohydrate interactions and structural characterization of Ralstonia solanacearum lectin
Antonik, Pawel
Antonik, Pawel
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2017-03-20
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Thesis
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Abstract
Ralstonia solanacearum is a bacterium which causes fatal damage to hundreds of agricultural plants such as potato or tobacco. It produces two lectins that are involved in attachment of the bacterium to the host. One of them is RSL that was recently characterized by X-ray crystallography. (1) Here, we present the first studies of RSL by NMR spectroscopy. The NMR assignments and dynamic studies of sugar-free and –bound RSL were the first milestone in our study of lectin-carbohydrate interactions in aqueous solutions (Chapter 2). (2) The NMR results provided valuable and unique information about anomeric-recognition effects that were not reported previously. This study also presented new insights that complement the RSL crystal structure. (1) The abundance of tryptophan residues provided useful probes in the HSQC spectrum. (1,2) With this broad background information we screened numerous carbohydrates and could interpret their binding events using CSP (Chapter 3). The recent interest in noncovalent protein-polymer conjugates sparked our imagination to test the concept with RSL. Reversible, noncovalent PEGylation of RSL using fucosylated PEG was demonstrated and characterized by NMR spectroscopy, size exclusion chromatography and native gel electrophoresis. A structural model of the RSL conjugate with fucosylated PEG was obtained by small-angle X-ray scattering experiments. Using ITC the affinity of the complex was assessed (Chapter 4). (3) In the last part of this thesis we demonstrate studies of RSL in physiological and crowded environments. In cell NMR spectroscopy was not eligible to obtain a spectrum of RSL in native conditions. Instead, intra-cellular mimicry was achieved using protein crowders, gelatin and agarose gels. Using NMR spectroscopy, significant differences in the interactions of sugar-free and -bound forms of RSL were demonstrated. The studies were supported by native agarose gel electrophoresis and SDS-PAGE revealing the aspect of confinement related to pore size (Chapter 5). (1) Kostlanova et al. J. Biol. Chem. 2005, 280, 27839–27849 (2) Antonik et al. Biochemistry. 2016, 55,1195–1203 (3) Antonik et al. Biomacromolecules 2016, 17, 2719–2725
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Attribution-NonCommercial-NoDerivs 3.0 Ireland