Control of subunit stoichiometry in single-chain MspA nanopores.
Control of subunit stoichiometry in single-chain MspA nanopores.
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单链 MspA 纳米孔中亚基化学计量的控制。
DOI：
10.1016/j.bpj.2022.01.022
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发表时间：
2022
影响因子：
3.4
通讯作者：
Niederweis,Michael
中科院分区：
文献类型：
--
作者：
Pavlenok,Mikhail;Yu,Luning;Herrmann,Dominik;Wanunu,Meni;Niederweis,Michael
跨膜蛋白通道能够快速、高灵敏地检测单分子。DNA的纳米孔测序是使用工程污垢分枝杆菌A(MSPA)和发动机酶相结合的方法实现的。由于其良好的通道几何结构，与其他孔蛋白相比，八聚体MSPA孔显示出最高的电流水平。到目前为止，MSPA是唯一一个公布了DNA测序记录的蛋白质纳米孔。虽然在商业设备中广泛使用，但由于复杂的DNA-马达-孔组合的随机事件以及每个位置最多五个核苷酸对信号的贡献，DNA纳米孔测序存在显著的碱基调用错误。孔蛋白特定亚基的不同突变为提高核苷酸分辨率和测序准确性提供了巨大的潜力。然而，MSPA的个别亚基和其他寡聚体蛋白质孔在体内和体外是随机组装的，阻碍了具有不同亚单位突变的设计孔的高效生产。在这项研究中，我们通过使用多肽连接物将八个亚基连接起来，将八聚体MSPA转化为单链孔。脂质双层实验证明，单链MSPA形成跨膜通道，并在DNA发夹实验中识别出与单体产生的MSPA相同的所有四个核苷酸。由三个、五个、六个和七个连接的亚基组成的单链结构组装成功能通道，展示了MSPA对不同亚基化学计量比的显著可塑性。因此，单链MSPA作为DNA测序和许多其他应用的生物传感器，通过产生具有不同亚单位突变和孔径的孔，构成了MSPA优化的一个新的里程碑。
Transmembrane protein channels enable fast and highly sensitive detection of single molecules. Nanopore sequencing of DNA was achieved using an engineeredMycobacterium smegmatisporin A (MspA) in combination with a motor enzyme. Due to its favorable channel geometry, the octameric MspA pore exhibits the highest current level compared with other pore proteins. To date, MspA is the only protein nanopore with a published record of DNA sequencing. While widely used in commercial devices, nanopore sequencing of DNA suffers from significant base-calling errors due to stochastic events of the complex DNA-motor-pore combination and the contribution of up to five nucleotides to the signal at each position. Different mutations in specific subunits of a pore protein offer an enormous potential to improve nucleotide resolution and sequencing accuracy. However, individual subunits of MspA and other oligomeric protein pores are randomly assembled in vivo and in vitro, preventing the efficient production of designed pores with different subunit mutations. In this study, we converted octameric MspA into a single-chain pore by connecting eight subunits using peptide linkers. Lipid bilayer experiments demonstrated that single-chain MspA formed membrane-spanning channels and discriminated all four nucleotides identical to MspA produced from monomers in DNA hairpin experiments. Single-chain constructs comprising three, five, six, and seven connected subunits assembled to functional channels, demonstrating a remarkable plasticity of MspA to different subunit stoichiometries. Thus, single-chain MspA constitutes a new milestone in the optimization of MspA as a biosensor for DNA sequencing and many other applications by enabling the production of pores with distinct subunit mutations and pore diameters.