Douette Pierre, Navet Rachel, Bouillenne Fabrice, Brans Alain, Sluse-Goffart Claudine, Matagne André, Sluse Francis E
Laboratory of Bioenergetics, Centre for Oxygen Research and Development, Institute of Chemistry B6, University of Liege, Sart Tilman, B-4000 Liege, Belgium.
Biochem J. 2004 May 15;380(Pt 1):139-45. doi: 10.1042/BJ20031957.
The rat UCP1 (uncoupling protein 1) is a mitochondrial inner-membrane carrier involved in energy dissipation and heat production. We expressed UCP1 carrying a His6 epitope at its C-terminus in Saccharomyces cerevisiae mitochondria. The recombinant-tagged UCP1 was purified by immobilized metal-ion affinity chromatography to homogeneity (>95%). This made it suitable for subsequent biophysical characterization. Fluorescence resonance energy transfer experiments showed that n-dodecyl-beta-D-maltoside-solubilized UCP1-His6 retained its PN (purine nucleotide)-binding capacity. The far-UV CD spectrum of the functional protein clearly indicated the predominance of alpha-helices in the UCP1 secondary structure. The UCP1 secondary structure exhibited an alpha-helical degree of approx. 68%, which is at least 25% higher than the previously reported estimations based on computational predictions. Moreover, the helical content remained unchanged in free and PN-loaded UCP1. A homology model of the first repeat of UCP1, built on the basis of X-ray-solved close parent, the ADP/ATP carrier, strengthened the CD experimental results. Our experimental and computational results indicate that (i) alpha-helices are the major component of UCP1 secondary structure; (ii) PN-binding mechanism does not involve significant secondary-structure rearrangement; and (iii) UCP1 shares similar secondary-structure characteristics with the ADP/ATP carrier, at least for the first repeat.
大鼠解偶联蛋白1（UCP1）是一种参与能量耗散和产热的线粒体内膜载体。我们在酿酒酵母线粒体中表达了在其C末端带有His6表位的UCP1。通过固定金属离子亲和层析将重组标记的UCP1纯化至同质（>95%）。这使其适用于后续的生物物理表征。荧光共振能量转移实验表明，用正十二烷基-β-D-麦芽糖苷增溶的UCP1-His6保留了其嘌呤核苷酸（PN）结合能力。功能蛋白的远紫外圆二色光谱清楚地表明UCP1二级结构中α螺旋占主导。UCP1二级结构的α螺旋度约为68%，这比先前基于计算预测的估计值至少高25%。此外，在游离的和PN负载的UCP1中，螺旋含量保持不变。基于X射线解析的亲缘关系较近的ADP/ATP载体构建的UCP1第一个重复序列的同源模型强化了圆二色实验结果。我们的实验和计算结果表明：（i）α螺旋是UCP1二级结构的主要成分；（ii）PN结合机制不涉及显著的二级结构重排；（iii）UCP1与ADP/ATP载体具有相似的二级结构特征，至少对于第一个重复序列是这样。
