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- All Subjects: Carrier proteins
- Creators: Allen, James
Description
Glycosaminoglycans (GAGs) are a class of complex biomolecules comprised of linear, sulfated polysaccharides whose presence on cell surfaces and in the extracellular matrix involve them in many physiological phenomena as well as in interactions with pathogenic microbes. Decorin binding protein A (DBPA), a Borrelia surface lipoprotein involved in the infectivity of Lyme disease, is responsible for binding GAGs found on decorin, a small proteoglycan present in the extracellular matrix. Different DBPA strains have notable sequence heterogeneity that results in varying levels of GAG-binding affinity. In this dissertation, the structures and GAG-binding mechanisms for three strains of DBPA (B31 and N40 DBPAs from B. burgdorferi and PBr DBPA from B. garinii) are studied to determine why each strain has a different affinity for GAGs. These three strains have similar topologies consisting of five α-helices held together by a hydrophobic core as well as two long flexible segments: a linker between helices one and two and a C-terminal tail. This structural arrangement facilitates the formation of a basic pocket below the flexible linker which is the primary GAG-binding epitope. However, this GAG-binding site can be occluded by the flexible linker, which makes the linker a negative regulator of GAG-binding. ITC and NMR titrations provide KD values that show PBr DBPA binds GAGs with higher affinity than B31 and N40 DBPAs, while N40 binds with the lowest affinity of the three. Work in this thesis demonstrates that much of the discrepancies seen in GAG affinities of the three DBPAs can be explained by the amino acid composition and conformation of the linker. Mutagenesis studies show that B31 DBPA overcomes the pocket obstruction with the BXBB motif in its linker while PBr DBPA has a retracted linker that exposes the basic pocket as well as a secondary GAG-binding site. N40 DBPA, however, does not have any evolutionary modifications to its structure to enhance GAG binding which explains its lower affinity for GAGs. GMSA and ELISA assays, along with NMR PRE experiments, confirm that structural changes in the linker do affect GAG-binding and, as a result, the linker is responsible for regulating GAG affinity.
ContributorsMorgan, Ashli M (Author) / Wang, Xu (Thesis advisor) / Allen, James (Committee member) / Yarger, Jeffery (Committee member) / Arizona State University (Publisher)
Created2015
Description
A novel small metal-binding protein (SmbP), with only 93 residues and no similarity to other known proteins, has been isolated from the periplasm of Nitrosomonas europaea. It is characterized by its high percentage (17%) of histidines, a motif of ten repeats of seven residues, a four α-helix bundle structure, and a high binding affinity to about six equivalents of Cu2+. The goal of this study is to investigate the Cu2+ binding sites in SmbP and to understand how Cu2+ stabilizes the protein. Preliminary folding experiments indicated that Cu2+ greatly stabilizes SmbP. In this study, protein folding data from circular dichroism (CD) spectroscopy was used to elucidate the role of Cu2+ in stabilizing SmbP structure against unfolding induced by decreased pH, increased temperature, and chemical denaturants. The significant stabilization effects of Cu2+ were demonstrated by the observation that Cu2+-SmbP remained fully folded under extreme environmental conditions, such as acidic pH, 96 °C, and 8 M urea. Also, it was shown that Cu2+ is able to induce the refolding of unfolded SmbP in acidic solutions. These findings imply that the coordination of Cu2+ to histidine residues is responsible for the stabilization effects. The crystal structure of SmbP without Cu2+ has been determined. However, attempts to crystallize Cu2+-SmbP have not been successful. In this study, multidimensional NMR experiments were conducted in order to gain additional information regarding the Cu2+-SmbP structure, in particular its metal binding sites. Unambiguous resonance assignments were successfully made. Cα secondary chemical shifts confirmed that SmbP has a four α-helical structure. A Cu2+-protein titration experiment monitored by NMR indicated a top-to-bottom, sequential metal binding pattern for SmbP. In addition, several bioinformatics tools were used to complement the experimental approach and identity of the ligands in Cu2+-binding sites in SmbP is proposed.
ContributorsYan, Qin (Author) / Francisco, Wilson A (Thesis advisor) / Allen, James (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2010