Pore-forming properties of proteolytically nicked staphylococcal ⋅-toxin: the ion channel in planar lipid bilayer membranes

Abstract

Staphylococcal alpha-toxin is a single-chain protein with a molecular mass of 33.2 kDa, which can form large water-filled pores both in lipid bilayers and in erythrocyte membranes. Limited proteolysis of the purified toxin with proteinase K led to time-dependent changes of all the functional features of the channels formed by the toxin. Single-channel conductance in planar bilayers was decreased about threefold. The anion selectivity of the channel was replaced with cation selectivity and the asymmetry in the current-voltage relationship of the channel became more pronounced. At the same time the nicked toxin kept its full ability to form ion channels in lipid bilayers, although it lost a considerable part of its hemolytic activity. In planar bilayers and in erythrocyte membranes, the proteolytically nicked toxin actually formed channels with a slightly smaller diameter (approximately 1.2 times) than that formed by the native toxin. This decrease was not marked enough to explain changes in the biological effects of the nicked toxin. The change in channel selectivity induced by the cleavage is considered to be the major determinant of the changes in the biological effects of the nicked toxin.

Ion channels and bacterial infection: the case of β‐barrel pore‐forming protein toxins of Staphylococcus aureus

“…2 ). Similar entrance diameters were also deduced from low‐resolution EM images [55,56], or atomic force microscopy [43,44], or from the differential permeability of sugar molecules of different sizes through pores reconstituted in artificial membranes [57]. In the EM, leukotoxin lesions appear remarkably similar to α‐hemolysin, with an entrance diameter between 20 and 30 Å [46,47].…”

Polymeric Nonelectrolytes to Probe Pore Geometry: Application to the α-Toxin Transmembrane Channel

“…In polymer-probing experiments the attraction has to be seen as F Ͼ 1.0, especially in the presence of PEG at both sides of the membrane. Indeed, such an effect was repeatedly reported by several groups (e.g., Krasilnikov et al, 1997;Bezrukov et al, 1996).…”

“…The most frequent value was observed in the range of 80 -120 pS. The small lower pools could be a reflection of noncomplete opening of the channel or/and slight (invisible in Coomassie-stained 10% polyacrylamide gels run in the presence of sodium dodecyl sulfate; data not shown) contamination of nicked toxin, which usually forms a low-conductance channel (Krasilnikov et al, 1997). These low-conductance pools were ignored and were not analyzed further.…”

Polymer Partitioning from Nonideal Solutions into Protein Voids

“…Distinctly separate populations of small conductances can be tentatively attributed to contaminations by nicked hemolysin. 32 Figure 3A summarizes our data on polymer-induced conductance reduction. It demonstrates that the interference of polymers with ionic current and, therefore, polymer partitioning depends on both polymer molecular weight and polymer concentration.…”

“…Therefore, the Gaussian-like spread of channel conductances is not related to the accuracy of our measurements (in the presence or absence of PEG) but rather to some minor deviations in the pore structure from channel to channel. Distinctly separate populations of small conductances can be tentatively attributed to contaminations by nicked hemolysin …”