De novo design, synthesis, and characterization of quinoproteins
De novo design, synthesis, and characterization of quinoproteins
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DOI：
10.1002/chem.200501212
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发表时间：
2006-09-18
影响因子：
4.3
通讯作者：
Haehnel, Wolfgang
中科院分区：
文献类型：
--
作者：
Li, Wen-Wu;Hellwig, Petra;Haehnel, Wolfgang
醌和醌蛋白是生物系统中重要的氧化还原组分和酶。在这里，我们报告的从头设计，合成和模型四α-螺旋束quinoproteins的属性。通过化学选择性连接到环状十肽模板，从具有21或22个氨基酸残基的三个不同螺旋设计和构建蛋白质。游离半胱氨酸单元位于蛋白质的疏水核心，用于通过硫醚键结合泛醌-0和甲萘醌-0。通过电喷雾质谱、紫外/维斯光谱、体积排阻色谱、圆二色谱、1H-1 NMR光谱、循环伏安法和氧化还原诱导FTIR差谱对分子量为11-12 kDa的醌蛋白进行了表征。对于泛醌-0和甲基萘醌-0，具有N-乙酰基半胱氨酸甲酯的硫醚缀合物的E-8相对于标准氢电极（SHE）分别为89 mV和-63 mV，并且具有合成蛋白质的E-8相对于标准氢电极（SHE）分别为229 mV和249 mV。对模型化合物和醌蛋白的详细的氧化还原诱导FTIR差光谱研究表明，由于硫分别取代为泛醌-0和甲基萘醌-0，在1656-1660和1655-1665 cm（-1）处的C=O带具有特殊的共振特征。模型quinoproteins的构建代表了向更复杂的人工氧化还原系统迈出的重要一步。
Quinones and quinoproteins are essential redox components and enzymes in biological systems. Here, we report the de novo design, synthesis, and properties of model four-alpha-helix bundle quinoproteins. The proteins were designed and constructed from three different helices with 21 or 22 amino acid residues by chemoselective ligation to a cyclic decapeptide template. A free cysteine unit is placed at the hydrophobic core of the protein for binding of ubiquinone-0 and menaquinone-0 through a thioether bond. The quinoproteins with molecular weights of 11-12 kDa were characterized by electrospray ionization mass spectrometry, UV/Vis spectroscopy, size-exclusion chromatography, circular dichroism measurements, H-1 NMR spectroscopy, cyclic voltammetry, and redox-induced FTIR difference spectroscopy The midpoint redox potentials at pH 8 in aqueous solution E-m,E-8 of thioether conjugates with N-acetyl cysteine methyl ester were 89 mV and -63 mV and with a synthetic protein 229 mV and 249 mV versus standard hydrogen electrode (SHE) for ubiquinone-0 and menaquinone-0, respectively. Detailed redox-induced FTIR difference spectroscopic studies, of the model compounds and quinoproteins show the special resonance features for C=O bands at 1656-1660 and 1655-1665 cm(-1) due to the sulfur substitution to ubiquinone-0 and menaquinone-0, respectively. The construction of model quinoproteins represents a significant step toward more complex artificial redox systems.