| Summary: | 碩士 === 國立成功大學 === 生物化學研究所 === 98 === Streptococcus pyogenes (group A Streptococcus [GAS]) is one of the most common human bacterial pathogens. GAS causes a variety of human diseases, including pharyngitis, impetigo, scarlet fever, cellulitis, toxic shock syndrome, necrotizing fasciitis. The genomes of many GAS strains have been sequenced, and they contained ~2000 genes. However, the functions of ~33% GAS genes are still unknown. In particular, most of these unknown genes are bacteriophage and transposon genes. It has been known that phage infection can enhance bacterial virulence, including bacterial adhesion, colonization, invasion; resistance to immune defenses; exotoxin production; sensitivity to antibiotics; and transmissibility among humans. In this study we identified a phage-associated hypothetical protein, SPy0985, which contains 88 amino acids residues with a superantigen signature sequence at the position of 49-75. SPy0985 was successfully expressed in E. coli and purified to homogeneity with a yield of 30 mg/L. We also found that of SPy0985 in the presence of 50 mM of arginine and glutamate (R+E) was properly folded with less aggregation. Molecular weight determination by size-exclusion chromatography analysis showed that SPy0985 existed as a shape of dimeric size under R+E and physiological conditions. The result of peripheral blood mononuclear cell assay showed that SPy0985 was a superantigen. NMR analysis showed that SPy0985 exhibited stable structures at N- and C-terminal regions with positive NOE and consisted of two α-helices packed against each other at C-terminus. It also formed a hydrophobic core packed by F8, Y17, Y23, L31 and Y36 at N terminal region. NMR relaxation studied showed that residues G39-D51 exhibited low NOE values, indicating the formation of a flexible loop in this region. Interestingly, the N-terminus and the loop ranging from K3 to T46 exhibited two resonances in 2D 1H-15N HSQC spectrum, indicating a chemical exchange process found from this region. The average NOE value of Spy0985 was 0.58, showing that SPy0985 was highly flexible. The R2/R1 ratio indicated that SPy0985 existed as a monomer. The underlying functional and structural details of SPy0985 remain to be elucidated.
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