Analysis of the secondary structure of the cys-less yeast mitochondrial citrate transport protein and four single-cys variants by circular dichroism.
Analysis of the secondary structure of the cys-less yeast mitochondrial citrate transport protein and four single-cys variants by circular dichroism.
复制标题
通过圆二色性分析无 cys 酵母线粒体柠檬酸转运蛋白和四种单 cys 变体的二级结构。
DOI：
10.1023/b:jobb.0000047325.48943.71
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发表时间：
2004
影响因子：
3
通讯作者：
Kaplan,RonaldS
中科院分区：
文献类型：
--
作者：
Cascio,Michael;Mayor,JuneA;Kaplan,RonaldS
利用半胱氨酸扫描诱变，与功能Cys柠檬酸转运蛋白（CTP）作为起始模板，我们以前证明，四个单Cys突变体位于跨膜结构域III和IV，使CTP非功能。本研究通过圆二色性（CD）光谱法评估和量化了无Cys CTP和四种单Cys突变体（在柠檬酸盐存在和不存在的情况下）的二级结构。在洗涤剂胶束中，高度纯化的Cys-less CTP含有约50%的α-螺旋和约20%的β-折叠。洗涤剂胶束中G119 C、E122 C、R181 C和R189 C突变体的CD光谱几乎与功能性Cys-少CTP的CD光谱重叠，从而表明野生型残基，而不是影响结构，可能承担重要的机械作用。外源性添加的柠檬酸盐引起所有溶解的CTP样品的CD光谱的显著变化。无Cys CTP的光谱分析表明，在柠檬酸盐存在下，其α-螺旋含量增加了约10%。通过添加底物的影响的构象变化是不太明显的单半胱氨酸突变体。在脂质体中重构的无Cys CTP的研究表明，虽然CD光谱发生红移，但重构载体的净二级结构与洗涤剂胶束中的转运蛋白的净二级结构大致相当，并显示出对添加的柠檬酸盐的响应。总之，上述研究表明，在肌氨酰胶束或脂质体中的纯化的无Cys CTP以及肌氨酰胶束中的四种无活性单Cys突变体保留天然样结构，因此代表了用于详细结构表征的理想材料。
Utilizing cysteine scanning mutagenesis, with functional Cys-less citrate transport protein (CTP) serving as the starting template, we previously demonstrated that four single-Cys mutants located in transmembrane domains III and IV, rendered the CTP nonfunctional. The present investigations assess and quantify the secondary structure of the Cys-less CTP and the four single-Cys mutants, both in the absence and presence of citrate, via circular dichroism (CD) spectroscopy. In detergent micelles, highly purified Cys-less CTP contained ∼50% α-helix and ∼20% β-sheet. The CD spectra of the G119C, E122C, R181C, and R189C mutants in detergent micelles were virtually superimposable with that of the functional Cys-less CTP, thereby suggesting that the wild-type residues, rather than affecting structure, may assume important mechanistic roles. Exogenously added citrate caused a significant change in the CD spectra of all solubilized CTP samples. Analyses of the spectra of the Cys-less CTP indicated an ∼10% increase in its α-helical content in the presence of citrate. The conformational changes effected by the addition of substrate were less pronounced with the single-Cys mutants. Studies of the Cys-less CTP reconstituted in liposomes indicated that while the CD spectra was red-shifted, the net secondary structure of the reconstituted carrier is approximately equivalent to that of the transporter in detergent micelles, and displayed a response to added citrate. In combination, the above studies indicate that purified Cys-less CTP in either sarkosyl micelles or in liposomes, and the four inactive single-Cys mutants in sarkosyl micelles, retain native-like structure, and thus represent ideal material for detailed structural characterization.