2008
DOI: 10.1016/j.jmb.2008.07.053
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Calorimetric Scrutiny of Lipid Binding by Sticholysin II Toxin Mutants

Abstract: The mechanisms by which pore-forming toxins are able to insert into lipid membranes are a subject of the highest interest in the field of lipid-protein interaction. Eight mutants affecting different regions of sticholysin II, a member of the pore-forming actinoporins family, have been produced and their hemolytic and lipidbinding properties compared to those of the wild-type protein. A thermodynamical approach to the mechanism of pore formation is also presented. Isothermal titration calorimetry experiments sh… Show more

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Cited by 52 publications
(137 citation statements)
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“…As we and others have previously proposed, there are several membrane-bound forms of EqtII at the membrane, and the increase in the NBD fluorescence reflects the two forms: one where helix lies more or less parallel to the membrane (M 2 in Fig. 7) (8,20,25) and the other one in the pore form (P), which may further change fluorescent properties of the NBD dye due to additional helix rearrangements. By the use of three different probes, we were therefore able to resolve three different forms of the protein at the surface of the lipid membrane (Fig.…”
Section: Discussionmentioning
confidence: 59%
See 1 more Smart Citation
“…As we and others have previously proposed, there are several membrane-bound forms of EqtII at the membrane, and the increase in the NBD fluorescence reflects the two forms: one where helix lies more or less parallel to the membrane (M 2 in Fig. 7) (8,20,25) and the other one in the pore form (P), which may further change fluorescent properties of the NBD dye due to additional helix rearrangements. By the use of three different probes, we were therefore able to resolve three different forms of the protein at the surface of the lipid membrane (Fig.…”
Section: Discussionmentioning
confidence: 59%
“…Actinoporins form cation-selective pores with a diameter of ϳ2 nm, mostly on membranes containing sphingomyelin (SM) (14 -16). The initial membrane attachment is achieved by an exposed aromatic cluster situated on the broad loop on the bottom of the molecule and the C-terminal ␣-helix and by a phosphorylcholine binding site (11,(17)(18)(19)(20)(21). The N-terminal region, residues 1-28, was shown to be the only part of the molecule that undergoes a conformational change, and its flexibility is essential for the formation of the final pore (19,(22)(23)(24).…”
mentioning
confidence: 99%
“…Disuccinimidyl suberate (DSS) was purchased from Pierce (Thermo Scientific). The preparation of the cDNA coding for StnI, StnII, and the six His-tagged version of StnII (6HStnII), as well as the production and purification of the three different proteins, has been described before (30,50,54). Homogeneity of all protein samples used was analyzed by 0.1% (w/v) SDS-12-15% PAGE (w/v) performed under standard conditions (55) and amino acid analysis after acid hydrolysis of the proteins (5.7 M HCl, 24 h, 110°C).…”
Section: Methodsmentioning
confidence: 99%
“…Both factors influence the conformational changes occurring during the transition from the water media to the inserted states of the protein (30,31). Thus, high affinity recognition of sphingomyelin (SM) 3 is crucial for specific attachment to a membrane, but the subsequent effects observed also depend on the physical properties derived from its particular composition and not only from its SM content (23,32).…”
mentioning
confidence: 99%
“…The binding isotherms were fitted to a model in which a molecule of protein binds to n molecules of lipid with the software ORIGIN 7 as described in Ref. 43. Each titration was repeated at least twice.…”
Section: Methodsmentioning
confidence: 99%