Award Date

8-2011

Degree Type

Thesis

Degree Name

Master of Science in Chemistry

Department

Chemistry

First Committee Member

Ernesto Abel-Santos, Chair

Second Committee Member

Dong-Chan Lee

Third Committee Member

Ronald Gary

Graduate Faculty Representative

Helen Wing

Number of Pages

81

Abstract

Bacillus anthracis,a gram positive, endospore-forming, rod-shaped, aerobic bacterium, is the causative agent of anthrax. A key step in the pathogenic cycle is the spore germination, where the dormant spore transforms into a metabolically active cell. This allows B. anthracis to proliferate and secrete toxins. It is believed that spore germination is triggered when small molecules, known as germinants, are recognized by germination (Ger) receptor proteins located in the inner membrane of spores. Most Ger proteins are encoded by tricistronic operons, resulting in three distinct products, the A-, B-, C-subunits. Genomic sequencing showed that B. anthracis has seven ger receptor operons- six genomic ger receptor operons (gerA, gerH, gerK, gerL, gerS, and gerY) and one plasmidic ger receptor operon (gerX).

The most efficient germinant combination is mixtures of L-alanine and purine nucleosides. 6-thioguanosine (6-TG), an inosine analog, was shown to be a competitive inhibitor of inosine mediated B. anthracis spore germination. 6-TG is a mercaptopurine, can be activated by UV irradiation to crosslink with binding proteins. By utilizing the properties of this compound, we aimed to identify Ger receptor proteins of B. anthracis.

To investigate purine binding site of the Ger proteins, we used a chemical-biological strategy. With the help of synthetic chemistry, 5'-azido-5'-deoxy-6-thioguanosine (N3 -6-TG) was prepared. Azide (-N3 ) functionality is introduced as a bait for click reaction and a thiol group (-SH) allows crosslinking. This compound was subsequently UV irradiated for crosslinking B. anthracis spores. After decoating and lysis of the spores, "click chemistry" was involved, expecting efficient acivity-based protein profiling with biotinylated alkyne tags under Cu(2)-catalyzed conditions. SDS-PAGE and detection with streptavidin-horseradish peroxidase conjugate were carried out to isolate the cross-linked proteins conjugated with the biotin tag. The detected area of SDS-PAGE gel can be excised and characterized by liquid chromatography-tandem mass spectrometry (LC-MS/ MS) analysis.

Keywords

Bacillus anthracis; Biochemistry; Chemical probe; Germination—Research; Protein profiling; Pure sciences; Spore germination; Thioguanosine

Disciplines

Biochemistry | Chemistry

Language

English


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