Regulations of export and chain length of extracellular bacterial polysaccharides
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Many Gram-positive and Gram-negative bacteria produce an additional thick layer of carbohydrate polymers on the cell wall surface. These capsules (capsular polysaccharides; CPS) play critical roles in interactions between bacteria and their environments (Whitfield, 2006). This is especially important in infection processes since for both Gram-negative and Gram-positive pathogens CPS is the point of first contact with the host immune system (Whitfield, 2006). However, the details of CPS biosynthesis and assembly mechanisms are still unclear. Therefore, we embarked on structural and kinetic studies of the proteins Wzc, Wza and Wzb/ Cps4B from the Wzy-dependent pathway, as well as the protein WbdD from the ATP-binding cassette (ABC) transporter dependent system. Full-length Wzc failed to crystallise due to the presence of large disordered regions and the overall difficulty of membrane protein crystallisation. A truncated version of Wzc (1-480) without the C-terminal tyrosine kinase domain was crystallised and diffracted to 15 Å in house. A previous study suggested Wza and Wzc form a functional complex (Whitfield, 2006), so Wza was also studied. Since the full-length Wza structure is available (C. Dong et al., 2006), Pulsed electron–electron double resonance spectroscopy (PELDOR) was used to study the conformational change. The PELDOR spectroscopy distance fingerprint of Wza was determined. These data also confirmed that PELDOR is a powerful tool to study large, highly symmetrical membrane proteins and can be used to study other complex membrane protein systems, such as ion channels or transporters. The crystal structure of Wzb the cognate phosphatase of Wzc was determined to 2.2 Å. Also Cps4B, which is a functional homologue of Wzb but has a completely unrelated sequence, was crystallised in two crystal forms. Form I and II Cps4B crystals diffracted to 2.8 Å and 1.9 Å resolution in house, respectively. The full-length WbdD failed to crystallise due to the presence of large disordered regions. Therefore, a shorter construct, WbdD₅₅₆ (1-556) was cloned and crystallised. The structure was determined to 2.2 Å. WbdD is a bifunctional enzyme consisting of a methyltransferase (MTase) and a kinase domain. In order to better understand the function of this protein, a variety of techniques were used, such as the ADP-Glo kinase assay, Nuclear magnetic resonance (NMR) spectroscopy, small angle X-ray scattering (SAXS) and X-ray crystallography. The various findings in the current projects provide meaningful insights towards a better understanding of the CPS biosynthesis and assembly mechanisms, which may contribute to a more intensive study identifying inhibitors and beginning to unravel the mechanism of chain length regulation.
Thesis, PhD Doctor of Philosophy
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