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A.G. Camacho, R. Misselwitz, J. Behlke, S. Ayora, K. Welfle, A. Meinhart, B. Lara, W. Saenger, H. Welfle, J.C. Alons
In vitro and in vivo Stability of the 2?2 Protein Complex of the Broad Host-Range Streptococcus pyogenes pSM19035 Addiction System
Streptococcus pyogenes pSM19035-encoded (10.7 kDa) and ? (32.4 kDa) proteins are necessary to secure stable plasmid inheritance in bacteria, with ? acting as toxin that kills plasmiddeprived cells and as an antitoxin that neutralises the activity of ?. The and ? proteins copurify as a stable complex that, according to analytical ultracentrifugation and gel filtration, exists as 2?2 heterotetramer in solution. Cocrystals of the 2?2 complex contain and ? in 1:1 molar ratio. Unfolding studies monitoring circular dichroic and fluorescence changes show that the ? protein has a significantly lower thermodynamic stability than the protein both in free state and in the complex. Proteolytic studies indicate that ? protein is more stable in the the 2?2 complex than in the free state. In vivo studies reveal a short halflife of the antitoxin (~18min) and a long lifetime of the ? toxin (>60min). When transcriptiontranslation of a plasmid containing the and ? genes was inhibited, cell death was observed after a short lag phase that correlates with the disappearance of the protein from the background.
Biological Chemistry, Walter de Gruyter
Print ISSN: 1431-6730
Volume: 383, 11/2002
Pages: 1701 - 1713