Fourier Transform Infrared Spectroscopy Study of the Secondary and Tertiary Structure of the Reconstituted Na+/Ca2+ Exchanger 70-kDa Polypeptide*
Fourier Transform Infrared Spectroscopy Study of the Secondary and Tertiary Structure of the Reconstituted Na+/Ca2+ Exchanger 70-kDa Polypeptide*
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DOI：
10.1074/jbc.274.22.15510
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发表时间：
1999-05
期刊：
影响因子：
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通讯作者：
R. I. Saba;J. Ruysschaert;A. Herchuelz;E. Goormaghtigh
中科院分区：
文献类型：
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作者：
R. I. Saba;J. Ruysschaert;A. Herchuelz;E. Goormaghtigh
用傅里叶变换红外衰减全反射光谱研究了纯化的70 kDa蛋白Na+/Ca~(2+)交换器的二级结构，并将其功能重组为asolectin脂泡。傅里叶变换红外衰减全反射光谱表明，该蛋白质由44%的α-螺旋、25%的β-折叠、16%的β-转数和15%的随机结构组成，值得注意的是，α-螺旋的比例高于交换分子亲水性分布预测的跨膜结构域的比例。偏光红外光谱分析表明，螺旋的取向几乎垂直于膜。通过对重组型交换器的氢/氢交换动力学测试，评价了由于添加钙离子而引起的三级结构的改变。这种方法以前被证明是一种有用的工具，用于检测蛋白质与其特定配体之间的相互作用引起的三级结构改变。氘/氢交换动力学测量表明，在没有钙离子的情况下，很大一部分(40%)的蛋白质是不能被溶剂访问的。Ca~(2+)的加入增加了对溶剂的不可及性的55%，这是一个主要的构象变化，其特征是至少屏蔽了93个氨基酸。
The secondary structure of the purified 70-kDa protein Na+/Ca2+ exchanger, functionally reconstituted into asolectin lipid vesicles, was examined by Fourier transform infrared attenuated total reflection spectroscopy. Fourier transform infrared attenuated total reflection spectroscopy provided evidence that the protein is composed of 44% α-helices, 25% β-sheets, 16% β-turns, and 15% random structures, notably the proportion of α-helices is greater than that corresponding to the transmembrane domains predicted by exchanger hydropathy profile. Polarized infrared spectroscopy showed that the orientation of helices is almost perpendicular to the membrane. Tertiary structure modifications, induced by addition of Ca2+, were evaluated by deuterium/hydrogen exchange kinetic measurements for the reconstituted exchanger. This approach was previously proven as a useful tool for detection of tertiary structure modifications induced by an interaction between a protein and its specific ligand. Deuterium/hydrogen exchange kinetic measurements indicated that, in the absence of Ca2+, a large fraction of the protein (40%) is inaccessible to solvent. Addition of Ca2+ increased to 55% the inaccessibility to solvent, representing a major conformational change characterized by the shielding of at least 93 amino acids.