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The deletion of several amino acid stretches of Escherichia coli alpha-hemolysin (HlyA) suggests that the channel-forming domain contains beta-strands.

Benz R, Maier E, Bauer S, Ludwig A - PLoS ONE (2014)

Bottom Line: These deletions resulted in HlyA mutants with strongly reduced hemolytic activity.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.

View Article: PubMed Central - PubMed

Affiliation: School of Engineering and Science, Jacobs University Bremen, Bremen, Germany.

ABSTRACT
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.

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Single-channel recordings with E. coli HlyA, HlyAΔ71–110, and HlyAΔ264–286.Single-channel recordings of asolectin membranes were performed in the presence of 50 ng/ml HlyA (left side, upper trace), 50 ng/ml HlyAΔ71–110 (left side, lower trace), and 50 ng/ml HlyAΔ264–286 (right side). The aqueous phase contained 150 mM KCl (pH 6). The applied membrane potential was 20 mV; T = 20°C. The average single-channel conductance was 520 pS for HlyA, 150 pS for HlyAΔ71–110, and 320 pS for HlyAΔ264–286.
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pone-0112248-g003: Single-channel recordings with E. coli HlyA, HlyAΔ71–110, and HlyAΔ264–286.Single-channel recordings of asolectin membranes were performed in the presence of 50 ng/ml HlyA (left side, upper trace), 50 ng/ml HlyAΔ71–110 (left side, lower trace), and 50 ng/ml HlyAΔ264–286 (right side). The aqueous phase contained 150 mM KCl (pH 6). The applied membrane potential was 20 mV; T = 20°C. The average single-channel conductance was 520 pS for HlyA, 150 pS for HlyAΔ71–110, and 320 pS for HlyAΔ264–286.

Mentions: We performed single-channel experiments with all four HlyA mutants. The experiments revealed that HlyAΔ71–110 and HlyAΔ264–286 still formed defined channels in lipid bilayer membranes but with much lower amplitude (i.e. single-channel conductance) than wildtype HlyA under otherwise identical conditions. Fig. 3 shows single-channel recordings of asolectin/n-decane membranes in the presence of wildtype HlyA, HlyAΔ71–110, and HlyAΔ264–286 in 150 mM KCl. All proteins were added to black membranes in a concentration of about 50 ng/ml. After a delay of several minutes, probably caused by slow aqueous diffusion and/or rearrangement of the toxin, we observed for HlyA and the two mutants the occurrence of transient ion-permeable channels. This means that the channels formed by HlyAΔ71–110 and HlyAΔ264–286 also had a limited lifetime (mean lifetime about 4 s) similar to wildtype HlyA [28]. Wildtype HlyA channels had a single-channel conductance, G, of about 520 pS, whereas the channels formed by HlyAΔ71–110 and HlyAΔ264–286 had with about 150 pS and 320 pS, respectively, a much smaller one (all in 150 mM KCl and at 20 mV membrane potential). The channel-forming activity was approximately the same for HlyA, HlyAΔ71–110, and HlyAΔ264–286, which means that at the same toxin concentration approximately the same number of channels was observed irrespective whether culture supernatants, precipitated proteins or pure toxins were added to the aqueous phase. The results thus indicated that these two mutations had only little influence on channel-forming probability or channel lifetime and affected only the single-channel conductance (i.e. the size) of the HlyA channel.


The deletion of several amino acid stretches of Escherichia coli alpha-hemolysin (HlyA) suggests that the channel-forming domain contains beta-strands.

Benz R, Maier E, Bauer S, Ludwig A - PLoS ONE (2014)

Single-channel recordings with E. coli HlyA, HlyAΔ71–110, and HlyAΔ264–286.Single-channel recordings of asolectin membranes were performed in the presence of 50 ng/ml HlyA (left side, upper trace), 50 ng/ml HlyAΔ71–110 (left side, lower trace), and 50 ng/ml HlyAΔ264–286 (right side). The aqueous phase contained 150 mM KCl (pH 6). The applied membrane potential was 20 mV; T = 20°C. The average single-channel conductance was 520 pS for HlyA, 150 pS for HlyAΔ71–110, and 320 pS for HlyAΔ264–286.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4251834&req=5

pone-0112248-g003: Single-channel recordings with E. coli HlyA, HlyAΔ71–110, and HlyAΔ264–286.Single-channel recordings of asolectin membranes were performed in the presence of 50 ng/ml HlyA (left side, upper trace), 50 ng/ml HlyAΔ71–110 (left side, lower trace), and 50 ng/ml HlyAΔ264–286 (right side). The aqueous phase contained 150 mM KCl (pH 6). The applied membrane potential was 20 mV; T = 20°C. The average single-channel conductance was 520 pS for HlyA, 150 pS for HlyAΔ71–110, and 320 pS for HlyAΔ264–286.
Mentions: We performed single-channel experiments with all four HlyA mutants. The experiments revealed that HlyAΔ71–110 and HlyAΔ264–286 still formed defined channels in lipid bilayer membranes but with much lower amplitude (i.e. single-channel conductance) than wildtype HlyA under otherwise identical conditions. Fig. 3 shows single-channel recordings of asolectin/n-decane membranes in the presence of wildtype HlyA, HlyAΔ71–110, and HlyAΔ264–286 in 150 mM KCl. All proteins were added to black membranes in a concentration of about 50 ng/ml. After a delay of several minutes, probably caused by slow aqueous diffusion and/or rearrangement of the toxin, we observed for HlyA and the two mutants the occurrence of transient ion-permeable channels. This means that the channels formed by HlyAΔ71–110 and HlyAΔ264–286 also had a limited lifetime (mean lifetime about 4 s) similar to wildtype HlyA [28]. Wildtype HlyA channels had a single-channel conductance, G, of about 520 pS, whereas the channels formed by HlyAΔ71–110 and HlyAΔ264–286 had with about 150 pS and 320 pS, respectively, a much smaller one (all in 150 mM KCl and at 20 mV membrane potential). The channel-forming activity was approximately the same for HlyA, HlyAΔ71–110, and HlyAΔ264–286, which means that at the same toxin concentration approximately the same number of channels was observed irrespective whether culture supernatants, precipitated proteins or pure toxins were added to the aqueous phase. The results thus indicated that these two mutations had only little influence on channel-forming probability or channel lifetime and affected only the single-channel conductance (i.e. the size) of the HlyA channel.

Bottom Line: These deletions resulted in HlyA mutants with strongly reduced hemolytic activity.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.

View Article: PubMed Central - PubMed

Affiliation: School of Engineering and Science, Jacobs University Bremen, Bremen, Germany.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus