Biophysics Department, Institute of Biology, University of Stuttgart, Germany.
J Membr Biol. 2011 Jul;242(1):11-21. doi: 10.1007/s00232-011-9372-8. Epub 2011 Jun 30.
Tom40 proteins represent an essential class of molecules which facilitate translocation of unfolded proteins from the cytosol into the mitochondrial intermembrane space. They are part of a high-molecular mass complex that forms the protein-conducting channel in outer mitochondrial membranes. This study concerns the recombinant expression, purification and folding of amino-terminally truncated variants of the two human Tom40 isoforms for structural biology experiments. Both CD and FTIR secondary structure analysis revealed a dominant beta-sheet structure and a short alpha-helical part for both proteins together with a high thermal stability. Two secondary structure elements can be denatured independently. Reconstitution of the recombinant protein into planar lipid bilayers demonstrated ion channel activity similar to Tom40 purified from Neurospora crassa mitochondrial membranes, but conductivity fingerprints differ from the structurally closely related VDAC proteins.
Tom40 蛋白是一类重要的分子，它们促进未折叠蛋白从细胞质易位到线粒体膜间隙。它们是形成线粒体外膜蛋白导通道的高分子质量复合物的一部分。本研究涉及两种人源 Tom40 同工型氨基末端截断变体的重组表达、纯化和折叠，用于结构生物学实验。CD 和 FTIR 二级结构分析显示，两种蛋白质都具有占主导地位的β-折叠结构和较短的α-螺旋部分，热稳定性高。两个二级结构元件可以独立变性。将重组蛋白重构到平面脂质双层中，证明了离子通道活性类似于从粗糙脉孢菌线粒体膜中纯化的 Tom40，但电导率指纹图谱与结构上密切相关的 VDAC 蛋白不同。
