Global redesign of a native β-barrel scaffold.
Global redesign of a native β-barrel scaffold.
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DOI：
10.1016/j.bbamem.2015.10.006
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发表时间：
2016-01
期刊：
影响因子：
--
通讯作者：
Movileanu L
中科院分区：
文献类型：
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作者：
Wolfe AJ;Mohammad MM;Thakur AK;Movileanu L
膜蛋白设计的一个长期挑战是在不损害其功能的情况下完成对蛋白质的广泛修饰。铁羟酸摄取成分A （FhuA）的截断衍生物，其特征是删除了160个残基软木结构域和5个大的细胞外环，产生了一个不导电的单体22链β桶蛋白转化为一个大导蛋白孔。在这里，我们发现这种重新设计的β-桶蛋白可以耐受内部表面电荷的广泛改变，包括25个负电荷中和。通过使用单分子电生理学，我们注意到各种截断的FhuA蛋白孔的共同点是在非常高的跨膜电位下发生33%的单一电流阻塞。我们确定这些电流转变是由它们与外部阳离子多肽的相互作用刺激的，这种相互作用以一种依赖于孔内部表面电荷以及多肽特征的方式发生。这项研究显示了在分子生物医学诊断、治疗和生物传感器技术中广泛工程的大单体β桶蛋白孔的前景。
One persistent challenge in membrane protein design is accomplishing extensive modifications of proteins without impairing their functionality. A truncation derivative of the ferric hydroxamate uptake component A (FhuA), which featured the deletion of the 160-residue cork domain and five large extracellular loops, produced the conversion of a non-conductive, monomeric, 22-stranded β-barrel protein into a large-conductance protein pore. Here, we show that this redesigned β-barrel protein tolerates an extensive alteration in the internal surface charge, encompassing 25 negative charge neutralizations. By using single-molecule electrophysiology, we noted that a commonality of various truncation FhuA protein pores was the occurrence of 33% blockades of the unitary current at very high transmembrane potentials. We determined that these current transitions were stimulated by their interaction with an external cationic polypeptide, which occurred in a fashion dependent on the surface charge of the pore interior as well as the polypeptide characteristics. This study shows promise for extensive engineering of a large monomeric β-barrel protein pore in molecular biomedical diagnosis, therapeutics, and biosensor technology.