Abstract
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.
Highlights
Despite the importance of membrane damaging proteins such as the immune proteins C9 of complement and perforin or pore-forming toxins from pathogenic bacteria, little is known about the precise mechanism that enables these proteins to convert from a water-soluble protein to a transmembrane channel
Certain amino acids near the C terminus were shown to be accessible to the hydrophobic probe 2-[3H]diazofluorene suggesting that they might penetrate into the membrane [20], but this remains to be confirmed using complementary methods
We have investigated whether the tryptophan residues in ␣-toxin reached the vicinity of the hydrophobic core of the bilayer upon membrane interaction
Summary
Despite the importance of membrane damaging proteins such as the immune proteins C9 of complement and perforin or pore-forming toxins from pathogenic bacteria, little is known about the precise mechanism that enables these proteins to convert from a water-soluble protein to a transmembrane channel. Using single cysteine mutants labeled with an environment-sensitive probe, Valeva and colleagues [16] have recently narrowed down the membrane inserting sequence to amino acids 118 –124 and suggested that these residues might line the channel. By the contribution of each monomer in the heptamer, the transmembrane domain of ␣-toxin would be a 14-stranded -barrel resembling the barrel of a porin monomer [17, 18] The existence of this barrel has recently been confirmed by x-ray crystallography [19]. It is unclear at the present time whether parts of the protein other than the central loop penetrate into the membrane. Tryptophan fluorescence spectroscopy, circular dichroism, and protease sensitivity were used to further compare the structure of the soluble, the membranebound, and the membrane-inserted forms of ␣-toxin
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