| Regional Biophysics Meeting 2005, March 16-20, Zreče, Slovenia | [ProtBiophys] |
Staphyloccoccus aureus produces gamma-hemolysins, toxins that are naturally cysteineless, produced as water-soluble monomers that assemble into oligomeric pores. Pore opening requires toxin oligomerisation into a non-lytic prepore and insertion of a beta-barrel into the lipid. The transmembrane portion is the stem region, in which each protomer contributes a beta-hairpin. These strands, folded onto the core of the protein in water, are extended through the membrane. This portion undergoes the main conformational changes required for pore formation. The dynamic of stem insertion is the less understood step. Stem unfolding is temperature dependent and can be arrested at 4°C on RBC, whereby hemolysis ensues if the cells are warmed up. To investigate this mechanism we have produced 4 double cys mutants of HlgB and their hemolytic activity was measured. Cys were introduced one at a common position, in the stretch that links the hairpin to the core, and one in a variable position along one of the strands. The distance between the cys was chosen to be bigger than that required for formation of cys-cys bonds, both in the water soluble and in the transmembrane form. Accordingly, at r.t., the mutants show leucocytolytic and hemolytic activity as the wt. However, they could be blocked in a prepore state by stimulating cys-cys bond formation with an oxidant. Prepore formation was obtained raising the temperature , but they remain non hemolytic: the pore-formation process is blocked. The extent of block was dependent on the dose of toxin and oxidant, and was removed by DTT. These results demonstrate that the unfolding of the stem can be interrupted at 4°C by the formation of cys-cys bonds, suggesting that during the extension process, all the positions mutated along the strand pass near enough to the fixed one to form a disulphide bond. °This study is based on an idea of him, and we like to have his name in the author list, even though he tragically passed away in July 2004.
Email: viero@itc.it
Address: Gabriella Viero- CNR-ITC Istituto di Biofisica Sezione di Trento- 38060 POVO (Italy)