MspA nanopores from subunit dimers.
MspA nanopores from subunit dimers.
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DOI：
10.1371/journal.pone.0038726
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发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Niederweis M
中科院分区：
文献类型：
--
作者：
Pavlenok M;Derrington IM;Gundlach JH;Niederweis M
耻垢分枝杆菌孔蛋白 A (MspA) 形成八聚体通道，是孔蛋白新家族的创始成员。亚基化学计量的控制对于为纳米技术应用定制 MspA 非常重要。在这项研究中，两个 MspA 单体通过长度为 17 至 62 个氨基酸的接头连接。从耻垢分枝杆菌中纯化出寡聚孔蛋白，并在脂质双层实验中显示出可形成功能通道。这些结果表明肽接头不会阻止 MspA 的正确折叠和定位。然而，与野生型MspA相比，表达水平降低了10倍。 MspA 因其独特的孔几何形状和稳健性而成为纳米孔测序的理想选择。为了评估由二聚体亚基制成的 MspA 在 DNA 测序中的有用性，我们连接了两个 M1-MspA 单体，其收缩区经过修饰以实现 DNA 易位。脂质双层实验证明该构建体也形成功能通道。由 M1 单体和 M1-M1 二聚体制成的 MspA 孔的电压门控相同，表明结构和动态通道特性相似。通过表达二聚体 mspA M1 基因，缺乏孔蛋白的耻垢分枝杆菌细胞中的葡萄糖摄取得以恢复，表明 M1-M1 孔在其天然膜中的正确折叠和定位。单链 DNA 发夹在由单体和亚基二聚体制成的孔中产生相同的离子电流阻断，证明 M1-M1 孔适合 DNA 测序。这项研究提供了原理证明，证明在耻垢分枝杆菌中产生单链 MspA 孔是可行的，并为产生具有改变的化学计量的 MspA 孔铺平了道路。亚基二聚体能够更好地控制 MspA 收缩区的化学和物理性质。这种方法对于理解分枝杆菌外膜转运和定制 MspA 用于 DNA 纳米孔测序都很有价值。
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.
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影响因子：
4.8
作者：
Hammerstein, Anne F.;Jayasinghe, Lakmal;Bayley, Hagan
通讯作者：
Bayley, Hagan
影响因子：
2.9
作者：
Nassi, S;Collier, RJ;Finkelstein, A
通讯作者：
Finkelstein, A
DOI：
10.1016/s1570-0232(03)00130-2
发表时间：
2003-06-25
影响因子：
3
作者：
Heinz, C;Karosi, S;Niederweis, M
通讯作者：
Niederweis, M
影响因子：
3.2
作者：
Guo, Xinzheng V.;Monteleone, Mercedes;Schnappinger, Dirk
通讯作者：
Schnappinger, Dirk
DOI：
10.1093/protein/8.7.725
发表时间：
1995-07-01
期刊：
PROTEIN ENGINEERING
影响因子：
--
作者：
ALFTHAN, K;TAKKINEN, K;TERRI, TT
通讯作者：
TERRI, TT