Departments of Chemistry, Wilfrid Laurier University, Waterloo, Ontario N2L 3C5, Canada.
Biochemistry. 2010 Jan 26;49(3):512-21. doi: 10.1021/bi901742g.
Mitochondrial uncoupling proteins of the nervous system (UCPs 2, 4, and 5) have potential roles in the function and protection of the central nervous system (CNS). In the absence of structural information, conformations of the hexahistidine-tagged versions of all five human UCPs in liposomes were investigated for the first time, using far- and near-UV CD and fluorescence spectroscopy. Highly pure UCPs 1-5 were reconstituted in detergents and stable small unilamellar vesicles, appropriate for spectroscopic studies. All UCPs formed dominantly helical conformations in negatively charged phospholipid vesicles (palmitoyloleoylphosphatidylcholine/palmitoyloleoylphosphatidylglycerol, 7:3 molar ratio). UCPs 2 and 5 exhibited comparable helical conformations with possible association in lipid bilayers, whereas UCP4 had a different helical profile that can be related to its less associated form. Interaction of reconstituted UCPs with GDP and GTP, inhibitors of the prototypic UCP1, was detected by near-UV CD and fluorescence spectroscopy, utilizing the sensitivity of these techniques to microenvironments around Trp residues close to the nucleotide binding site. Binding of UCP4 to purine nucleotides was also different from other UCPs. Binding of fatty acids, activators of proton transport in UCPs, to UCPs could not be unambiguously detected, implying a nonbinding conformation/orientation of the proteoliposomes. Interaction of CoA with UCPs was comparable to nucleotide binding, suggesting a possible binding of this molecule at the nucleotide binding site. Despite dissimilar primary sequences, neuronal UCPs share common structural and functional properties with UCPs 1 and 3, supporting a common physiological role in addition to their specific roles in the CNS.
神经系统的线粒体解偶联蛋白（UCPs 2、4 和 5）在中枢神经系统（CNS）的功能和保护中具有潜在作用。在缺乏结构信息的情况下，首次使用远紫外和近紫外 CD 以及荧光光谱法研究了脂质体中所有五种人 UCP 的六组氨酸标记版本的构象。高度纯的 UCPs 1-5 在去污剂和稳定的小单层囊泡中进行重组，适用于光谱研究。所有 UCPs 在带负电荷的磷脂囊泡（棕榈酰油酰磷脂酰胆碱/棕榈酰油酰磷脂酰甘油，7:3 摩尔比）中形成主要的螺旋构象。UCPs 2 和 5 表现出相似的螺旋构象，可能在脂质双层中存在缔合，而 UCP4 具有不同的螺旋轮廓，这可能与其较少缔合的形式有关。利用这些技术对靠近核苷酸结合位点的色氨酸残基周围微环境的敏感性，通过近紫外 CD 和荧光光谱法检测了 GDP 和 GTP 与重组 UCPs 的相互作用，抑制剂为原型 UCP1。UCP4 与嘌呤核苷酸的结合也与其他 UCPs 不同。脂肪酸与 UCPs 的结合，即 UCPs 中质子转运的激活剂，不能明确检测到，这意味着蛋白脂囊泡的结合构象/取向是非结合的。CoA 与 UCPs 的相互作用与核苷酸结合相当，这表明该分子可能在核苷酸结合位点结合。尽管初级序列不同，但神经元 UCPs 与 UCPs 1 和 3 具有共同的结构和功能特性，除了它们在 CNS 中的特定作用外，还支持其在生理上具有共同作用。
