Biochemical, stabilization and crystallization studies on a molecular chaperone (PaoD) involved in the maturation of molybdoenzymes.
Biochemical, stabilization and crystallization studies on a molecular chaperone (PaoD) involved in the maturation of molybdoenzymes.
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DOI：
10.1371/journal.pone.0087295
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Santos-Silva T
中科院分区：
文献类型：
--
作者：
Otrelo-Cardoso AR;Schwuchow V;Rodrigues D;Cabrita EJ;Leimkühler S;Romão MJ;Santos-Silva T
钼和钨酶需要特定的分子伴侣进行折叠和辅因子插入。PaoD是周质醛氧化还原酶PaoABC的伴侣。它是大肠杆菌中paoABCD操纵子中的最后一个基因，并且它的存在对于获得成熟酶至关重要。PaoD是一种不稳定的35 kDa蛋白质。我们的生化研究表明，它是一种二聚体在溶液中的倾向，形成大的聚集体，特别是在冻融循环。为了提高稳定性，PaoD在两种离子液体[C4 mim]Cl和[C2 OHmim] PF 6的存在下解冻，并且没有观察到蛋白质沉淀。这允许使用聚乙二醇或硫酸铵作为沉淀剂进行蛋白质浓缩和结晶。还进行了饱和转移差-核磁共振（STD-NMR）实验，以研究离子液体在稳定化过程中的作用，表明阳离子的酸性环质子与蛋白质表面的带负电荷的残基之间存在明显的相互作用。DLS测定还显示在离子液体存在下蛋白质聚集体的总体尺寸减小。此外，对PaoD的辅因子结合研究表明，该蛋白能够区分钼和钨结合到钼辅因子，因为只有钼-MPT形式的辅因子保持结合到PaoD。
Molybdenum and tungsten enzymes require specific chaperones for folding and cofactor insertion. PaoD is the chaperone of the periplasmic aldehyde oxidoreductase PaoABC. It is the last gene in the paoABCD operon in Escherichia coli and its presence is crucial for obtaining mature enzyme. PaoD is an unstable, 35 kDa, protein. Our biochemical studies showed that it is a dimer in solution with a tendency to form large aggregates, especially after freezing/thawing cycles. In order to improve stability, PaoD was thawed in the presence of two ionic liquids [C4mim]Cl and [C2OHmim]PF6 and no protein precipitation was observed. This allowed protein concentration and crystallization using polyethylene glycol or ammonium sulfate as precipitating agents. Saturation transfer difference – nuclear magnetic resonance (STD-NMR) experiments have also been performed in order to investigate the effect of the ionic liquids in the stabilization process, showing a clear interaction between the acidic ring protons of the cation and, most likely, negatively charged residues at the protein surface. DLS assays also show a reduction of the overall size of the protein aggregates in presence of ionic liquids. Furthermore, cofactor binding studies on PaoD showed that the protein is able to discriminate between molybdenum and tungsten bound to the molybdenum cofactor, since only a Mo-MPT form of the cofactor remained bound to PaoD.
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DOI：
10.1107/s0907444905036693
发表时间：
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影响因子：
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