Cascio Michael, Mayor June A, Kaplan Ronald S
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA.
J Bioenerg Biomembr. 2004 Oct;36(5):429-38. doi: 10.1023/B:JOBB.0000047325.48943.71.
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 approximately 50% alpha-helix and approximately 20% beta-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 approximately 10% increase in its alpha-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.
利用半胱氨酸扫描诱变技术，以无半胱氨酸的功能性柠檬酸转运蛋白（CTP）作为起始模板，我们之前证明位于跨膜结构域III和IV的四个单半胱氨酸突变体使CTP失去功能。本研究通过圆二色性（CD）光谱，评估并量化了无半胱氨酸的CTP和四个单半胱氨酸突变体在有无柠檬酸存在时的二级结构。在去污剂胶束中，高度纯化的无半胱氨酸CTP含有约50%的α-螺旋和约20%的β-折叠。去污剂胶束中G119C、E122C、R181C和R189C突变体的CD光谱与功能性无半胱氨酸CTP的光谱几乎完全重叠，从而表明野生型残基可能承担重要的机制作用，而非影响结构。外源添加柠檬酸导致所有溶解的CTP样品的CD光谱发生显著变化。对无半胱氨酸CTP光谱的分析表明，在有柠檬酸存在时其α-螺旋含量增加了约10%。单半胱氨酸突变体因添加底物而引起的构象变化不太明显。对脂质体中重构的无半胱氨酸CTP的研究表明，虽然CD光谱发生了红移，但重构载体的净二级结构与去污剂胶束中的转运体大致相当，并对添加的柠檬酸有响应。综合来看，上述研究表明，在十二烷基肌氨酸钠胶束或脂质体中的纯化无半胱氨酸CTP，以及十二烷基肌氨酸钠胶束中的四个无活性单半胱氨酸突变体，保留了类似天然的结构，因此是进行详细结构表征的理想材料。
