Structure analysis of the protein translocating channel TatA in membranes using a multi-construct approach
Structure analysis of the protein translocating channel TatA in membranes using a multi-construct approach
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DOI：
10.1016/j.bbamem.2007.06.021
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发表时间：
2007-10-01
影响因子：
3.4
通讯作者：
Ulrich, Anne S.
中科院分区：
文献类型：
--
作者：
Lange, Christian;Mueller, Sonja D.;Ulrich, Anne S.
双精氨酸移位酶（达特）可以将折叠状态的蛋白质运输穿过细菌或类囊体膜。在枯草芽孢杆菌中，Tat机制仅由两种完整的（内）膜蛋白TatA和TatC组成。TatA的多个拷贝应该形成跨膜通道，但是关于这个70个残基的组分的结构数据很少。我们使用多构建体方法来表达TatA（d）的几个特征片段，以确定它们的个体结构并在全长蛋白质的结构内全面交叉验证它们。在这里，我们报告的设计，高产量的表达，洗涤剂辅助的纯化和脂质重建的五个结构的TatA（d），克服困难与非常不同的疏水性和这些膜蛋白片段的大小。圆二色性（CD）和定向CD（OCD）被用来确定它们各自的构象和对齐在合适的，带负电荷的磷脂双层。CD光谱显示N-末端α-螺旋，中心螺旋拉伸和非结构化C-末端，从而首次证明TatA（d）中存在这些二级结构。OCD光谱表明N-末端α-螺旋的跨膜取向和脂质双层中中心两亲性螺旋的表面对齐，从而支持TatA作为跨膜通道的假定拓扑模型和功能。(C)2007 Elsevier B. V.保留所有权利。
The twin-arginine-translocase (Tat) can transport proteins in their folded state across bacterial or thylakoid membranes. In Bacillus subtilis the Tat-machinery consists of only two integral (inner) membrane proteins, TatA and TatC. Multiple copies of TatA are supposed to form the transmembrane channel, but little structural data is available on this 70-residue component. We used a multi-construct approach for expressing several characteristic fragments of TatA(d), to determine their individual structures and to cross-validate them comprehensively within the architecture of the full-length protein. Here, we report the design, high-yield expression, detergent-aided purification and lipid-reconstitution of five constructs of TatA(d), overcoming difficulties associated with the very different hydrophobicities and sizes of these membrane protein fragments. Circular dichroism (CD) and oriented CD (OCD) were used to determine their respective conformations and alignments in suitable, negatively charged phospholipid bilayers. CD spectroscopy showed an N-terminal alpha-helix, a central helical stretch, and an unstructured C-terminus, thus proving the existence of these secondary structures in TatA(d) for the first time. The OCD spectra demonstrated a transmembrane orientation of the N-terminal alpha-helix and a surface alignment of the central amphiphilic helix in lipid bilayers, thus supporting the postulated topology model and function of TatA as a transmembrane channel. (C) 2007 Elsevier B.V. All rights reserved.