Subunit Dimers of α-Hemolysin Expand the Engineering Toolbox for Protein Nanopores
Subunit Dimers of α-Hemolysin Expand the Engineering Toolbox for Protein Nanopores
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DOI：
10.1074/jbc.m111.218164
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发表时间：
2011-04-22
影响因子：
4.8
通讯作者：
Bayley, Hagan
中科院分区：
文献类型：
--
作者：
Hammerstein, Anne F.;Jayasinghe, Lakmal;Bayley, Hagan
葡萄球菌 α-溶血素 (α HL) 形成一个七聚体孔，其特征是 14 链跨膜 β-桶。我们试图通过串联基因的体外转录和翻译产生的亚基二聚体寡聚化来迫使αHL孔采用新的化学计量。然而，体外转录和翻译也产生了截短的蛋白质、单体，它们优先掺入寡聚物中。通过单通道记录显示这些寡聚体是功能性七聚体，并且在 SDS 聚丙烯酰胺凝胶中具有与野生型七聚体相似的迁移率。然后使用 His 标记的蛋白质制备纯化的全长亚基二聚体。单通道记录再次表明，由这些二聚体制成的寡聚体是功能性七聚体，这意味着一个或多个亚基被排除在中心孔之外。因此，α HL 孔可抵抗除那些紧邻中心轴的七个亚基之外的所有结构。尽管我们无法通过亚基串联来改变 α HL 中心孔的化学计量，但我们扩展了我们的发现，制备了包含一个亚基二聚体和五个单体的孔，并通过 SDS-PAGE 纯化了它们。然后将两个半螯合配体安装在相邻位点，二聚体的每个亚基上各一个。单通道记录表明，由该结构形成的孔与二价金属离子形成复合物，其方式与同一亚基上含有两个半螯合配体的孔类似，证实寡聚物已与中心腔周围的七个亚基组装在一起。将亚基二聚体整合到 α HL 孔中的能力增加了可以从工程蛋白质纳米孔中获得的结构范围。
Staphylococcal alpha-hemolysin (alpha HL) forms a heptameric pore that features a 14-stranded transmembrane beta-barrel. We attempted to force the alpha HL 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 alpha HL 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 alpha HL 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 alpha HL pores increases the range of structures that can be obtained from engineered protein nanopores.