The staphylococcal alpha-toxin pore has a flexible conformation

The alpha-toxin from Staphylococcus aureus undergoes several conformational changes from the time it is released from the bacterium to the moment it forms a channel in the plasma membrane of its target cell. It is initially a soluble monomer, which undergoes membrane binding and oligomerization into a heptameric ring and finally inserts into the lipid bilayer to form a pore. Here we have analyzed the stability of different forms of the alpha-toxin (monomer as well as heptamers in solution, bound to the membrane and membrane-inserted) by differential scanning calorimetry and limited proteolysis. Data presented here show that, in contrast to both the membrane-bound prepore complex and the monomer in solution, the membrane-inserted alpha-toxin channel does not undergo cooperative unfolding and is highly susceptible to proteases. These observations suggest that the channel has a looser conformation. Interestingly, resistance to proteases could be recovered upon solubilization of the channel, indicating that the loss of rigid tertiary packing only occurred upon membrane insertion. Far-UV CD data, however, suggest that the transmembrane beta-barrel must be stably folded and that therefore only the Cap and Rim domains of the channel are loosely packed. All together, our data show that the alpha-toxin channel is not a rigid complex within the membrane but adopts a rather flexible conformation

Conformational changes due to membrane binding and channel formation by staphylococcal alpha-toxin

Conformational changes occurring upon membrane binding and subsequent insertion of staphylococcal alpha-toxin were studied using complementary spectroscopic techniques. Experimental conditions were established where binding could be uncoupled from membrane insertion but insertion and channel formation seemed to be concomitant. Binding led to changes in tertiary structure as witnessed by an increase in tryptophan fluorescence, a red shift of the tryptophan maximum emission wavelength, and a change in the near UV CD spectrum. In contrast to what was observed for the soluble form of the toxin, 78% of the tryptophan residues in the membrane-bound form were accessible to the hydrophilic quencher KI. At this stage, the tryptophan residues were not in the immediate vicinity of the lipid bilayer. Upon membrane insertion, a second conformational change occurred resulting in a dramatic drop of the near UV CD signal but an increase of the far UV signal. Tryptophan residues were no longer accessible to KI but could be quenched by brominated lipids. In the light of the available data on channel formation by alpha-toxin, our results suggest that the tryptophan residues might be dipping into the membrane in order to anchor the extramembranous part of the channel to the lipid bilayer

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in the subunit stoichiometry study of high mass non-covalent complexes

This study explores the potential of MALDI-TOF MS for the mass measurement of large non-covalent protein complexes. The following non-covalent complexes have been investigated: aerolysin from Aeromonas hydrophila (335 kDa) and α-haemolysin from Staphylococcus aureus (233 kDa) which are both cytolytic toxins, three enzymes known to be homotetramers in solution: bovine liver catalase (235 kDa), rabbit muscle pyruvate kinase (232 kDa), yeast alcohol dehydrogenase (147 kDa) and finally a lectin, concanavalin A (102 kDa). Three different matrix preparations were systematically tested under various conditions: ferulic acid dissolved in THF, 2,6-dihydroxyacetophenone in 20 mM aqueous ammonium citrate and a two-step sample preparation with sinapinic acid. It was possible to find a suitable combination of matrix and preparation type which allowed the molecularity of all complexes tested to be deduced from the MALDI mass spectrum. Trimeric and tetrameric intermediates accumulating during the formation of the active heptameric aerolysin complex were also identified, this allowing a formation mechanism to be proposed. The observation of large specific non-covalent complexes has been found to be dependent on the choice of matrix, the type of sample preparation used, the solvent evaporation speed, the pH of the resulting matrix-sample mixture and the number of shots acquired on a given area. From this set of experiments, some useful guidelines for the observation of large complexes by MALDI could therefore be deduced. Fast evaporation of the solvent is particularly necessary in the case of pH sensitive complexes. An ESMS study on the same non-covalent complexes indicated that, rather surprisingly, reliable results could be obtained by MALDI-TOF MS on several very large complexes (above 200 kDa) for which ESMS yielded no clear spectra