Structural and Functional Studies of Regulatory Proteins in the Bacterial Exopolysaccharide Synthesis and Transport Pathways

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Chesterman, Chelsy Caryn
Regulatory Proteins , Lipopolysaccharide , Capsular Polysaccharide , Etk , LapB , Structural Biology
Extracellular sugar polymers, or exopolysaccharides, play crucial roles in gram-negative bacteria that enable their survival and proliferation in hostile environments. As a result, the proteins involved in the synthesis, transport, and regulation of these sugars are potential targets for antibiotic development. Escherichia coli tyrosine kinase (Etk) is an inner membrane protein that is part of the capsular polysaccharide (CPS) export complex. The autophosphorylation of Etk is considered to be the regulatory signal that controls CPS export, but the mechanism for transmitting this signal to the other members of the protein complex has yet to be elucidated. It is expected that the periplasmic domain of Etk is involved. The first portion of this thesis discusses progress towards determining an x-ray crystal structure for Etk’s periplasmic region. Significant advancement occurred when the unstable full-length Etk protein was truncated to include only the periplasmic domain and the two transmembrane helices. Attempts to improve the reproducibility and quality of crystals obtained provided the realization that detergent concentration was a crucial factor. This led to the development of an improved method for detergent concentration measurement in small-volume, membrane protein samples using a colorimetric reaction with 2,6-dimethylphenol. Other traditional chemical reactions that can be applied to detergent measurement were also tested. Another important exopolysaccharide produced in E. coli is lipopolysaccharide. The second part of this thesis presents the first crystal structure of a newly identified, key regulator of lipopolysaccharide synthesis, lipopolysaccharide assembly protein B (LapB). Zn-SAD phasing with high-redundancy data was used to determine the experimental phases required for structure solution. Retrospective analysis of the same data revealed that this structure could also have been solved with lower redundancy data. The protein contains nine tetratricopeptide repeats and a rubredoxin metal binding domain that fold together in an unexpectedly intimate arrangement. Proper functioning of LapB, including the binding of the rubredoxin domain to the TPR helices appears crucial for optimum cell growth.
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