Production and initial structural characterization of the TM4TM5 helix-loop-helix domain of the translocator protein
Production and initial structural characterization of the TM4TM5 helix-loop-helix domain of the translocator protein
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DOI：
10.1002/psc.2468
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发表时间：
2013-02-01
影响因子：
2.1
通讯作者：
Jamin, N.
中科院分区：
文献类型：
--
作者：
Galvagnion, C.;Montaville, P.;Jamin, N.
主要存在于线粒体中的转运蛋白TSPO，以前称为外周苯二氮卓受体，是一种小的必需膜蛋白，参与胆固醇跨线粒体膜的转运，这是类固醇生物合成中的速率决定步骤。我们以前报道了五个片段的结构，包括五个假定的跨膜螺旋，并表明这些片段中的每一个构成一个自主折叠单元。为了进一步表征负责这种膜蛋白的helixhelix协会的结构决定因素，我们现在调查的折叠双跨膜结构域在各种洗涤剂胶束。在此，我们提出了成功的生物合成的双跨膜结构域，包括最后两个C-末端螺旋（TM 4 TM 5）。为了在大肠杆菌中最佳地产生该结构域，需要评估各种肽构建体，包括与不同纯化标签或与增溶蛋白融合的TM 4 TM 5。报道了纯度大于95%的TM_4TM_5的生产工艺。使用圆二色性和溶液状态NMR进一步表征该结构域。远紫外圆二色性研究表明，TM 4 TM 5的二级结构是高度螺旋的，当溶解在各种洗涤剂胶束，包括正十二烷基-β-d-麦芽糖苷，正辛基-β-d-葡糖苷，正十二烷基磷酸胆碱，1，2-二己酰基-sn-甘油基-3-磷酸胆碱（DHPC），和1-棕榈酰基-2-羟基-sn-甘油基-3-磷酸-（1 '-rac-甘油）。此外，该结构域的增溶条件进行了优化的NMR实验，初步分析表明，TM 4 TM 5采用一个稳定的三级折叠内TM 4 TM 5-DHPC复合物。版权所有（c）2013 European Peptide Society and John Wiley & Sons，Ltd.
Mainly present in the mitochondria, the translocator protein, TSPO, previously known as the peripheral benzodiazepine receptor, is a small essential membrane protein, involved in the translocation of cholesterol across mitochondrial membranes, a rate determining step in steroids biosynthesis. We previously reported the structure of five fragments encompassing the five putative transmembrane helices and showed that each of these fragments constitutes an autonomous folding unit. To further characterize the structural determinants responsible for helixhelix association of this membrane protein, we now investigate the folding of double transmembrane domains in various detergent micelles. Herein, we present the successful biosynthesis of a double transmembrane domain encompassing the last two C-terminal helices (TM4TM5). For optimal production of this domain in Escherichia coli, the evaluation of various peptide constructs, including TM4TM5 fused to different purification tags or to solubilizing proteins, was necessary. The protocol of production of TM4TM5 with more than 95% purity is reported. This domain was further characterized using circular dichroism and solution state NMR. Far-UV circular dichroism studies indicate that the secondary structure of TM4TM5 is highly helical when solubilized in various detergent micelles including n-dodecyl-beta-d-maltoside, n-octyl-beta-d-glucoside, n-dodecylphosphocholine, 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC), and 1-palmitoyl-2-hydroxy-sn-glycero-3-phospho-(1'-rac-glycerol). In addition, the solubilization conditions of the domain were optimized for NMR experiments, and preliminary analysis indicates that TM4TM5 adopts a stable tertiary fold within the TM4TM5-DHPC complex. Copyright (c) 2013 European Peptide Society and John Wiley & Sons, Ltd.