Klingenberg M, Appel M
Institut für Physikalische Biochemie, Universität München, Federal Republic of Germany.
Eur J Biochem. 1989 Mar 1;180(1):123-31. doi: 10.1111/j.1432-1033.1989.tb14622.x.
Isolated uncoupling protein (UCP) can be cross-linked, by various disulfide-forming reagents, to dimers. The best cross-linking is achieved with Cu2+-phenanthroline oxidation. Because cross-linking is independent of UCP concentration and prevented by SDS addition, a disulfide bridge must be formed between the two subunits of the native dimer. Cross-linking is prevented by SH reagent and reversed by SH-reducing reagents. In mitochondria, cross-linking of UCP with disulfide-forming agents is even more efficient than in isolated state. It proves that UCP is a dimer in mitochondria, before isolation. Disulfide-bridge formation does not inhibit GTP-binding to UCP. Cross-linked UCP re-incorporated in proteoliposomes either before or after cross-linking fully retains the H1-transport function. Rapid cross-linking by membrane impermeant reagents indicates a surface localization of the C-terminus in soluble UCP and projection to the outer surface in mitochondria. Intermolecular disulfide-bridge formation in a dimer requires juxtaposition of identical cysteines at the twofold symmetry axis. A rigid juxtaposition of cysteines is unlikely, unless intended for a native disulfide bridge. The absence of such a bridge in UCP suggests that juxtaposition of cysteines is generated by high mobility. In order to localize the cysteine involved, cross-linked UCP was cleaved by BrCN. The CB-7 C-terminal peptide, which contains cysteines at positions 287 and 304, disappears. Limited trypsinolytic cleavage, previously shown to occur at Lys-292, removed cross-linking in UCP both in the solubilized and mitochondrially bound state. The cleaved C-terminal peptide of 11 residues contains only cystein-304 which, thus, should be the only one (out of 7 cysteines in UCP) involved in the S-S bridge formation. Obviously, the C-terminal location of the cysteine, because of its high mobility, permits juxtapositioning for cross-linking. This agrees with predictions from hydrophobicity analysis that the last 14 residues in UCP protrude from the membrane.
分离出的解偶联蛋白（UCP）可通过各种形成二硫键的试剂交联成二聚体。用Cu2 + -菲咯啉氧化可实现最佳交联。由于交联与UCP浓度无关且可被SDS添加所阻止，因此天然二聚体的两个亚基之间必定形成了二硫键。交联可被SH试剂阻止，并可被SH还原试剂逆转。在线粒体中，UCP与形成二硫键的试剂的交联比在分离状态下更有效。这证明在分离之前，UCP在线粒体中就是二聚体。二硫键的形成并不抑制GTP与UCP的结合。交联后的UCP在交联之前或之后重新掺入蛋白脂质体中，完全保留了H1转运功能。膜不透性试剂的快速交联表明C末端在可溶性UCP中的表面定位以及在线粒体中外表面的突出。二聚体中分子间二硫键的形成需要在二重对称轴上并列相同的半胱氨酸。除非是天然二硫键，否则半胱氨酸不太可能刚性并列。UCP中不存在这样的桥表明半胱氨酸的并列是由高迁移率产生的。为了定位所涉及的半胱氨酸，用溴化氰切割交联的UCP。包含第287位和第304位半胱氨酸的CB - 7 C末端肽消失。先前显示在Lys - 292处发生的有限胰蛋白酶切割消除了UCP在溶解状态和线粒体结合状态下的交联。切割后的11个残基的C末端肽仅包含半胱氨酸 - 304，因此，它应该是（UCP的7个半胱氨酸中）参与S - S桥形成的唯一半胱氨酸。显然，由于其高迁移率，半胱氨酸的C末端定位允许并列进行交联。这与疏水性分析的预测一致，即UCP中的最后14个残基从膜中突出。
