Folgering Joost H A, Wolters Justina C, Poolman Bert
Department of Biochemistry, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands.
Protein Sci. 2005 Dec;14(12):2947-54. doi: 10.1110/ps.051679005.
To obtain a gene construct for making single substitutions per channel and to determine the quaternary structure of the mechanosensitive channel MscL from Escherichia coli, covalent oligomers (monomer to hexamer) were engineered by gene fusion; up to six copies of the mscL gene were fused in tandem. All the multimeric tandem constructs yielded functional channels with wild-type conductance and dwell times. Importantly, only the covalent pentamer opened at the same relative pressure (compared to the pressure required to open MscS) as the wild-type MscL channel. The in vivo data strongly suggest that pentameric MscL represents the functional state of the channel.
为获得用于在每个通道进行单取代的基因构建体，并确定来自大肠杆菌的机械敏感通道MscL的四级结构，通过基因融合构建了共价寡聚体（单体至六聚体）；将多达六个拷贝的mscL基因串联融合。所有多聚体串联构建体均产生具有野生型电导和驻留时间的功能性通道。重要的是，只有共价五聚体在与野生型MscL通道相同的相对压力下（与打开MscS所需的压力相比）打开。体内数据有力地表明，五聚体MscL代表该通道的功能状态。
