MspA Nanopores from Subunit Dimers

Mycobacterium smegmatis porin A (MspA) forms an octameric channel and represents the founding member of a new family of pore proteins. Control of subunit stoichiometry is important to tailor MspA for nanotechnological applications. In this study, two MspA monomers were connected by linkers ranging from 17 to 62 amino acids in length. The oligomeric pore proteins were purified from M. smegmatis and were shown to form functional channels in lipid bilayer experiments. These results indicated that the peptide linkers did not prohibit correct folding and localization of MspA. However, expression levels were reduced by 10-fold compared to wild-type MspA. MspA is ideal for nanopore sequencing due to its unique pore geometry and its robustness. To assess the usefulness of MspA made from dimeric subunits for DNA sequencing, we linked two M1-MspA monomers, whose constriction zones were modified to enable DNA translocation. Lipid bilayer experiments demonstrated that this construct also formed functional channels. Voltage gating of MspA pores made from M1 monomers and M1-M1 dimers was identical indicating similar structural and dynamic channel properties. Glucose uptake in M. smegmatis cells lacking porins was restored by expressing the dimeric mspA M1 gene indicating correct folding and localization of M1-M1 pores in their native membrane. Single-stranded DNA hairpins produced identical ionic current blockades in pores made from monomers and subunit dimers demonstrating that M1-M1 pores are suitable for DNA sequencing. This study provides the proof of principle that production of single-chain MspA pores in M. smegmatis is feasible and paves the way for generating MspA pores with altered stoichiometries. Subunit dimers enable better control of the chemical and physical properties of the constriction zone of MspA. This approach will be valuable both in understanding transport across the outer membrane in mycobacteria and in tailoring MspA for nanopore sequencing of DNA.

耻垢分枝杆菌孔蛋白A（Mycobacterium smegmatis porin A, MspA）是一种八聚体通道蛋白，亦是孔蛋白家族新分支的首个代表性成员。调控亚基化学计量比对于将MspA适配纳米技术应用至关重要。本研究中，研究人员通过长度为17至62个氨基酸的连接肽将两个MspA单体相连。从耻垢分枝杆菌中纯化得到的寡聚孔蛋白，经脂质双层实验验证可形成功能性通道。实验结果表明，该连接肽并未阻碍MspA的正确折叠与定位。不过，与野生型MspA相比，其表达水平下降了10倍。MspA凭借其独特的孔道几何结构与稳定性，是纳米孔测序的理想选择。为评估由二聚化亚基构成的MspA在DNA测序中的应用价值，研究人员将两个M1-MspA单体进行了连接，该单体的收缩区已被改造以适配DNA转位。脂质双层实验证实，此构建体同样可形成功能性通道。由M1单体与M1-M1二聚体构成的MspA孔道的电压门控特性完全一致，表明二者具有相似的结构与动态通道属性。在缺失孔蛋白的耻垢分枝杆菌细胞中，表达二聚体形式的mspA M1基因可恢复葡萄糖摄取能力，这表明M1-M1孔道可在其天然膜中正确折叠并定位。单链DNA发夹结构在单体与二聚体亚基构成的孔道中产生了完全一致的离子电流阻断信号，证实M1-M1孔道适用于DNA测序。本研究从原理上证明了在耻垢分枝杆菌中生产单链MspA孔道具备可行性，为构建具有改变后化学计量比的MspA孔道铺平了道路。二聚化亚基可更好地调控MspA收缩区的理化性质。该研究方法不仅有助于理解分枝杆菌外膜的跨膜运输机制，还可用于适配MspA以实现DNA纳米孔测序。