Molecular dynamics simulations were performed to describe the function of the ion-channel-forming toxin α-hemolysin (αHL) in lipid membranes that were composed of either 1,2-diphytanoyl-sn-glycero-3-phospho-choline or 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-choline. The simulations highlight the importance of lipid type in maintaining αHL structure and function, enabling direct comparison to experiments for biosensing applications. We determined that although the two lipids studied are similar in structure, 1,2-diphytanoyl-sn-glycero-3-phospho-choline membranes better match the hydrophobic thickness of αHL compared to 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-choline membranes. This hydrophobic match is essential to maintaining proper alignment of β-sheet loops at the trans entrance of αHL, which, when disrupted, creates an additional constriction to ion flow that decreases the channel current below experimental values and creates greater variability in channel conductance. Agreement with experiments was further improved with sufficient lipid membrane equilibration and allowed the discrimination of subtle αHL conduction states with lipid type. Finally, we explore the effects of truncating the extramembrane cap of αHL and its role in maintaining proper alignment of αHL in the membrane and channel conductance. Our results demonstrate the essential role of lipid type and lipid-protein interactions in simulations of αHL and will considerably improve the interpretation of experimental data.
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