TY  - JOUR
A1  - Benz, Roland
A1  - Maier, Elke
A1  - Bauer, Susanne
A1  - Ludwig, Albrecht
T1  - The Deletion of Several Amino Acid Stretches of Escherichia coli Alpha-Hemolysin (HlyA) Suggests That the Channel-Forming Domain Contains Beta-Strands
JF  - PLOS ONE
N2  - Escherichia coli α-hemolysin (HlyA) is a pore-forming protein of 110 kDa belonging to the family of RTX toxins. A hydrophobic region between the amino acid residues 238 and 410 in the N-terminal half of HlyA has previously been suggested to form hydrophobic and/or amphipathic α-helices and has been shown to be important for hemolytic activity and pore formation in biological and artificial membranes. The structure of the HlyA transmembrane channel is, however, largely unknown. For further investigation of the channel structure, we deleted in HlyA different stretches of amino acids that could form amphipathic β-strands according to secondary structure predictions (residues 71–110, 158–167, 180–203, and 264–286). These deletions resulted in HlyA mutants with strongly reduced hemolytic activity. Lipid bilayer measurements demonstrated that HlyAΔ71–110 and HlyAΔ264–286 formed channels with much smaller single-channel conductance than wildtype HlyA, whereas their channel-forming activity was virtually as high as that of the wildtype toxin. HlyAΔ158–167 and HlyAΔ180–203 were unable to form defined channels in lipid bilayers. Calculations based on the single-channel data indicated that the channels generated by HlyAΔ71–110 and HlyAΔ264–286 had a smaller size (diameter about 1.4 to 1.8 nm) than wildtype HlyA channels (diameter about 2.0 to 2.6 nm), suggesting that in these mutants part of the channel-forming domain was removed. Osmotic protection experiments with erythrocytes confirmed that HlyA, HlyAΔ71–110, and HlyAΔ264–286 form defined transmembrane pores and suggested channel diameters that largely agreed with those estimated from the single-channel data. Taken together, these results suggest that the channel-forming domain of HlyA might contain β-strands, possibly in addition to α-helical structures.
KW  - membrane potential
KW  - molecular mass
KW  - cations
KW  - membrane structures
KW  - membrane proteins
KW  - lipid bilayer
KW  - red blood cells
KW  - toxins
Y1  - 2014
U6  - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-118115
SN  - 1932-6203
VL  - 9
IS  - 12
ER  -