Rial E, Muga A, Valpuesta J M, Arrondo J L, Goñi F M
Department of Biochemistry, Faculty of Science, University of the Basque Country, Bilbao, Spain.
Eur J Biochem. 1990 Feb 22;188(1):83-9. doi: 10.1111/j.1432-1033.1990.tb15374.x.
The uncoupling protein of brown-adipose-tissue mitochondria has been purified in the form of mixed micelles with lipid and reduced Triton X-100. This surfactant has the advantage over conventional Triton X-100, that it does not interfere with amide bands in infrared spectra. The structure of the uncoupling protein in micellar form has been examined by Fourier-transform infrared spectroscopy (FTIR). In order to decompose the amide I contour into its components, band-narrowing (Fourier derivation and deconvolution) and band-decomposition techniques have been used. Combining data from spectra taken in H2O and 2H2O media, the following percentage distribution of secondary structure patterns has been obtained: 50% alpha-helix, 28-30% beta-structure; 13-15% beta-turns and 7% unordered. Thermal denaturation of the uncoupling protein has also been monitored by FTIR. In accordance with previous observations of different proteins, thermal denaturation is marked by a shift in the amide I maximum and the appearance of two new peaks in 2H2O, at around 1620 cm-1 and 1685 cm-1. Denaturation occurs in the 40-50 degrees C temperature range, in agreement with studies of GDP-binding capacity. Cooling down the thermally denatured protein produces a new change in its secondary structure; however, the original conformation is not restored. The uncoupling protein possesses a nucleotide-binding site. On addition of GDP, small changes in protein conformation occur, attributable to changes in tertiary structure. However, no detectable effects are seen in the presence or absence of the other physiological regulators, the free fatty acids. The uncoupling protein shares important similarities in its primary structure with other anion carriers of the mitochondrial membrane; one of these, the adenine-nucleotide translocator, has been used in a comparative study, applying the same FTIR techniques described above for the uncoupling protein. Both proteins have a similar proportion of alpha-helix, probably corresponding to the segments spanning the membrane, but the conformation of the polar domains appears to differ.
棕色脂肪组织线粒体的解偶联蛋白已以与脂质和还原型 Triton X-100 形成混合胶束的形式被纯化。这种表面活性剂相对于传统的 Triton X-100 具有优势，即它不会干扰红外光谱中的酰胺带。已通过傅里叶变换红外光谱 (FTIR) 研究了胶束形式的解偶联蛋白的结构。为了将酰胺 I 轮廓分解为其组成部分，使用了谱带变窄（傅里叶微分和去卷积）和谱带分解技术。结合在 H2O 和 2H2O 介质中获得的光谱数据，得到了以下二级结构模式的百分比分布：50% 的 α-螺旋、28 - 30% 的 β-结构；13 - 15% 的 β-转角和 7% 的无规结构。解偶联蛋白的热变性也通过 FTIR 进行了监测。与先前对不同蛋白质的观察结果一致，热变性的特征是酰胺 I 最大值的移动以及在 2H2O 中出现两个新峰，分别在 1620 cm-1 和 1685 cm-1 左右。变性发生在 40 - 50 摄氏度的温度范围内，这与 GDP 结合能力的研究结果一致。将热变性的蛋白质冷却会使其二级结构产生新的变化；然而，原始构象并未恢复。解偶联蛋白具有一个核苷酸结合位点。加入 GDP 后，蛋白质构象会发生小的变化，这归因于三级结构的变化。然而，在存在或不存在其他生理调节剂游离脂肪酸的情况下，未观察到可检测到的影响。解偶联蛋白在其一级结构上与线粒体膜的其他阴离子载体具有重要的相似性；其中之一，腺嘌呤核苷酸转位酶，已被用于一项比较研究，采用了上述用于解偶联蛋白的相同 FTIR 技术。两种蛋白质的 α-螺旋比例相似，可能对应于跨膜片段，但极性结构域的构象似乎不同。
