Department of Biochemistry, School of Medical Sciences, University of Bristol, Bristol BS8 1TD, United Kingdom.
J Biol Chem. 2011 May 27;286(21):18807-15. doi: 10.1074/jbc.M110.176891. Epub 2011 Feb 23.
Studies on membrane protein folding have focused on monomeric α-helical proteins and a major challenge is to extend this work to larger oligomeric membrane proteins. Here, we study the Escherichia coli (E. coli) ATP-binding cassette (ABC) transporter that imports vitamin B(12) (the BtuCD protein) and use it as a model system for investigating the folding and assembly of a tetrameric membrane protein complex. Our work takes advantage of the modular organization of BtuCD, which consists of two transmembrane protein subunits, BtuC, and two cytoplasmically located nucleotide-binding protein subunits, BtuD. We show that the BtuCD transporter can be re-assembled from both prefolded and partly unfolded, urea denatured BtuC and BtuD subunits. The in vitro re-assembly leads to a BtuCD complex with the correct, native, BtuC and BtuD subunit stoichiometry. The highest rates of ATP hydrolysis were achieved for BtuCD re-assembled from partly unfolded subunits. This supports the idea of cooperative folding and assembly of the constituent protein subunits of the BtuCD transporter. BtuCD folding also provides an opportunity to investigate how a protein that contains both membrane-bound and aqueous subunits coordinates the folding requirements of the hydrophobic and hydrophilic subunits.
膜蛋白折叠的研究主要集中在单体 α-螺旋蛋白上，一个主要的挑战是将这项工作扩展到更大的寡聚膜蛋白上。在这里，我们研究了大肠杆菌（E. coli）ATP 结合盒（ABC）转运蛋白，该蛋白可导入维生素 B12（BtuCD 蛋白），并将其用作研究四聚体膜蛋白复合物折叠和组装的模型系统。我们的工作利用了 BtuCD 的模块化组织，它由两个跨膜蛋白亚基 BtuC 和两个位于细胞质中的核苷酸结合蛋白亚基 BtuD 组成。我们表明，BtuCD 转运蛋白可以从预折叠和部分变性的脲变性 BtuC 和 BtuD 亚基重新组装。体外重新组装导致具有正确的天然 BtuC 和 BtuD 亚基比例的 BtuCD 复合物。从部分变性的亚基重新组装的 BtuCD 获得了最高的 ATP 水解速率。这支持了 BtuCD 转运蛋白组成蛋白亚基的协同折叠和组装的观点。BtuCD 折叠还为研究包含膜结合和水相亚基的蛋白质如何协调疏水性和亲水性亚基的折叠要求提供了机会。
