Chemical Sciences - Master's Theses

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Browsing Chemical Sciences - Master's Theses by Subject "anthrax"

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    The influence of pH and chemical denaturants on metal binding and structure of anthrax lethal factor
    (2017-02-15) Lo, Becky Suet Yan
    Bacillus anthracis is the causative agent of anthrax, and secretes a three-component toxin consisting of protective antigen (PA), edema factor (EF), and lethal factor (LF). LF is a zinc-dependent metallopeptidase responsible for cleaving mitogen-activated protein kinase kinases in the cytosol. To reach the cytosol, LF must be unfolded to pass through the narrow lumen of the PA channel embedded in the endosomal membrane. Whether the metal-binding motif in LF remains intact, and the Zn2+ ion is co-translocated along with the protein is currently unknown. Using a combination of intrinsic tryptophan fluorescence spectroscopy and chelator studies to probe the unfolding and metal status of LF, this study shows that acidification from pH 7 to pH 5 leads a marked destabilization of LF’s native fold, and a significantly increased degree of Zn2+ accessibility (to chelation) and release. Furthermore, red-edge excitation shift studies show that LF still retains a partial fold even when exposed to low pH and high concentrations of denaturants. Taken together, these results provide insights into the structure and metal status of LF under conditions similar to those encountered during translocation in vivo, and they suggest that LF’s Zn2+ ion is likely lost during PA-mediated translocation.
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    Studies on metal exchange and inhibition in anthrax lethal factor
    (2016-06-15) Young, Calvin J.
    Anthrax lethal factor (LF) is a zinc-dependent endopeptidase, and a component of the anthrax toxin secreted by Bacillus anthracis. LF demonstrates two interesting contradictory features: a propensity to readily exchange the active site metal ion with extraneous metal ions in solution, while having at the same time a very high affinity (femto- to picomolar) for metals in its active site. The hypothesis of this thesis was that metal exchange in LF occurs via an associative mechanism involving the formation of a di-metal species through the occupation of an inhibitory metal-binding site (by the incoming metal) located adjacent to the active site. We demonstrate here that the spontaneous demetallation of LF is slow, especially for the release of zinc (t1/2 ~3 h). In contrast, metal exchange (with 70Zn2+, Co2+ and Cu2+) was found to occur with t1/2 values between 5 s and 180 s, hence supporting an associative mechanism of exchange. In addition, the mode of inhibition exerted by Zn2+ on zinc-containing LF (ZnLF) was observed to be non-competitive with an inhibition (pseudo)constant of ~30 μM, a feature in agreement with turnover studies using stopped-flow UV-Vis spectroscopy. Finally, present studies using Tb3+ suggest that the lanthanide ion competes with other metal ions (Zn2+, Cu2+) for binding to the inhibitory metal site in LF, hence decreasing the rate of metal exchange. This observation provides further support for the intimate link between a putative inhibitory binding site and metal exchange. The results of this study provide insight into how cellular metal pools may remain dynamic in vivo, and have implications not only for LF, but for many other zinc-dependent enzymes where metal substitution is used for spectroscopic studies.