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Abstract
Staphylococcal α-hemolysin (αHL) forms a heptameric pore that features a 14-stranded transmembrane β-barrel. We attempted to force the αHL pore to adopt novel stoichiometries by oligomerizing subunit dimers generated by in vitro transcription and translation of a tandem gene. However, in vitro transcription and translation also produced truncated proteins, monomers, that were preferentially incorporated into oligomers. These oligomers were shown to be functional heptamers by single-channel recording and had a similar mobility to wild-type heptamers in SDS-polyacrylamide gels. Purified full-length subunit dimers were then prepared by using His-tagged protein. Again, single-channel recording showed that oligomers made from these dimers are functional heptamers, implying that one or more subunits are excluded from the central pore. Therefore, the αHL pore resists all structures except those that possess seven subunits immediately surrounding the central axis. Although we were not able to change the stoichiometry of the central pore of αHL by the concatenation of subunits, we extended our findings to prepare pores containing one subunit dimer and five monomers and purified them by SDS-PAGE. Two half-chelating ligands were then installed at adjacent sites, one on each subunit of the dimer. Single-channel recording showed that pores formed from this construct formed complexes with divalent metal ions in a similar fashion to pores containing two half-chelating ligands on the same subunit, confirming that the oligomers had assembled with seven subunits around the central lumen. The ability to incorporate subunit dimers into αHL pores increases the range of structures that can be obtained from engineered protein nanopores.
Highlights
Introduction of Functionality across TwoSubunits of the ␣HL Pore—To demonstrate the utility of a subunit dimer in a heptameric pore, we introduced the half-chelating ligand propyliminodiacetic acid (PIDA) (Fig. 10A) on each of the two subunits within a dimer of a 2115714330 JOURNAL OF BIOLOGICAL CHEMISTRY pore
The oligomers were mixed with Laemmli sample buffer (1ϫ final concentration), and a portion was heated to 95 °C for 10 min before electrophoresis in a 10% BisTris polyacrylamide gel (Bio-Rad) in MOPS running buffer
Design and Expression of Subunit Dimers of ␣HL—The termini of the individual subunits in a concatemer are usually linked by short peptide sequences
Summary
(9), and the construction of “prototissues” based on droplets connected by bilayers [10, 11] To approach these goals, engineered ␣HL pores are essential, and they have been prepared by site-directed mutagenesis with natural and unnatural amino acids and by both noncovalent and covalent chemical modification [12, 13]. The protective antigen of anthrax toxin, which is structurally similar to the ␣HL pore but lacks sequence similarity, exists in both heptameric and octameric forms [20, 21] These results suggest that it might be possible to manipulate the subunit stoichiometry of the ␣HL pore, and this was attempted in this study. We failed to accomplish the first goal, the latter was successful
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