Inspection of the Engineered FhuA ΔC/Δ4L Protein Nanopore by Polymer Exclusion
Inspection of the Engineered FhuA ΔC/Δ4L Protein Nanopore by Polymer Exclusion
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DOI：
10.1016/j.bpj.2012.10.008
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发表时间：
2012-11-21
影响因子：
3.4
通讯作者：
Movileanu, Liviu
中科院分区：
文献类型：
--
作者：
Niedzwiecki, David J.;Mohammad, M. Mohammad;Movileanu, Liviu
使用天然支架对蛋白质纳米孔进行广泛的生物技术应用工程需要进一步检查其内部几何形状和尺寸。最近，我们重新设计了异羟肟酸铁吸收成分 A (FhuA)，这是一种含有 N 端 160 个残基 cork 结构域 (C) 的 22-β 链蛋白。删除软木结构域和四个大的细胞外环 (4L)，以获得异常坚硬的工程 FhuA Delta C/Delta 4L 纳米孔。我们使用水溶性聚乙二醇和葡聚糖聚合物来检查 Fhu Delta C/Delta 4L 的内部。当该纳米孔被重构为合成平面脂质双层时，聚乙二醇的添加产生了单通道电导的改变，从而可以评估纳米孔直径。在这里，我们报道 FhuA Delta C/Delta 4L 具有近似圆锥形的内部几何形状，顺式入口小于反式入口，这与野生型 FhuA 蛋白晶体结构的不对称性质一致。使用不可渗透的葡聚糖聚合物进行的进一步实验表明平均内径类似于 2.4 nm，这是我们根据聚合物诱导的进入阻力对纳米孔总阻力贡献的变化得出的结论。从这项工作中推断出的分子见解代表了 FhuA 未来蛋白质工程的平台，该平台将用于生物技术应用中的特定任务。
Extensive engineering of protein nanopores for biotechnological applications using native scaffolds requires further inspection of their internal geometry and size. Recently, we redesigned ferric hydroxamate uptake component A (FhuA), a 22-beta-stranded protein containing an N-terminal 160-residue cork domain (C). The cork domain and four large extracellular loops (4L) were deleted to obtain an unusually stiff engineered FhuA Delta C/Delta 4L nanopore. We employed water-soluble poly(ethylene glycols) and dextran polymers to examine the interior of Fhu Delta C/Delta 4L. When this nanopore was reconstituted into a synthetic planar lipid bilayer, addition of poly(ethylene glycols) produced modifications in the single-channel conductance, allowing for the evaluation of the nanopore diameter. Here, we report that FhuA Delta C/Delta 4L features an approximate conical internal geometry with the cis entrance smaller than the trans entrance, in accord with the asymmetric nature of the crystal structure of the wild-type FhuA protein. Further experiments with impermeable dextran polymers indicated an average internal diameter of similar to 2.4 nm, a conclusion we arrived at based upon the polymer-induced alteration of the access resistance contribution to the nanopore's total resistance. Molecular insights inferred from this work represent a platform for future protein engineering of FhuA that will be employed for specific tasks in biotechnological applications.