Modification of lysine residues of Staphylococcus aureus alpha-toxin: effects on its channel-forming properties.
Staphylococcus aureus alpha-toxin opens an ion channel in planar phospholipid bilayers, which is selective for anions over cations, supposedly because of the presence of positively charged groups along the ion pathway. To remove some positive charges of this protein toxin, we chemically modified part of its lysine residues either with diethylpyrocarbonate, followed by histidine regeneration with hydroxylamine, or with trinitrobenzenesulfonic acid. The extent of chemical modification can be followed accurately by native polyacrylamide gel electrophoresis and isoelectric focusing. Ethoxyformilation of two to three lysine residues per toxin monomer does not impair hemolysis of rabbit red blood cells nor formation of pores in model membranes. It reduces the conductance and the anion selectivity of the channel and changes the shape of its current-voltage characteristic. This indicates that positively charged lysine residues are actually important in determining the electrical properties of the pore. Ethoxyformilation of channels preassembled in planar bilayers produces the same changes as modification of toxin monomers before channel formation. Furthermore, it can be performed by adding diethylpyrocarbonate on either side of the bilayer. This suggests that the lysine residues relevant for the electrical properties of the pore are located inside its lumen where they can be reached by diethylpyrocarbonate diffusing from either entrance of the channel.
Cescatti
L
L
Dipartimento di Fisica, Università di Trento, Italy.
Pederzolli
C
C
Menestrina
G
G
D001427:Bacterial Toxins / Q000737:chemistry / Q000378:metabolism*; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology*; D004563:Electrochemistry; D004591:Electrophoresis, Polyacrylamide Gel; D006460:Hemolysin Proteins / Q000737:chemistry / Q000378:metabolism*; D006639:Histidine / Q000378:metabolism; D019811:Hydroxylamine; D006898:Hydroxylamines / Q000494:pharmacology; D007473:Ion Channels / Q000378:metabolism*; D007525:Isoelectric Focusing; D008051:Lipid Bilayers; D008239:Lysine / Q000378:metabolism*; D013211:Staphylococcus aureus / Q000378:metabolism*; D014302:Trinitrobenzenesulfonic Acid / Q000494:pharmacology
D001427:Bacterial Toxins; D006460:Hemolysin Proteins; D006898:Hydroxylamines; D007473:Ion Channels; D008051:Lipid Bilayers; C021091:staphylococcal alpha-toxin; D019811:Hydroxylamine; D006639:Histidine; D014302:Trinitrobenzenesulfonic Acid; D008239:Lysine; D004047:Diethyl Pyrocarbonate