Abstract
Many soluble and membrane proteins form symmetrical homooligomeric complexes. However, determining the oligomeric state of protein complexes can be difficult. Alpha-hemolysin (αHL) from Staphylococcus aureus is a symmetrical homooligomeric protein toxin that forms transmembrane β-barrel pores in host cell membranes. The stable pore structure of αHL has also been exploited in vitro as a nanopore tool. Early structural experiments suggested αHL forms a hexameric pore, while more recent X-ray crystal structure and solution studies have identified a heptameric pore structure. Here, using native ion mobility-mass spectrometry (IM-MS) we find that αHL simultaneously forms hexameric and heptameric oligomers in both tetraethylene glycol monooctyl ether (C(8)E(4)) and tetradecylphosphocholine (FOS-14) detergent solutions. We also analyze intact detergent micelle-embedded αHL porelike complexes by native IM-MS without the need to fully strip the detergent micelle, which can cause significant gas-phase unfolding. The highly congested native mass spectra are deconvolved using Fourier- and Gábor-transform (FT and GT) methods to determine charge states and detergent stoichiometry distributions. The intact αHL micelle complexes are found to contain oligomeric state-proportional numbers of detergent molecules. This evidence, combined with IM data and results from vacuum molecular dynamics simulations, is consistent with both the hexamer and the heptamer forming porelike complexes. The ability of αHL to form both oligomeric states simultaneously has implications for its use as a nanopore tool and its pore formation mechanism in vivo. This study also demonstrates more generally the power of FT and GT to deconvolve the charge state and stoichiometry distributions of polydisperse ions.