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Solubilization and Purification of the MotA/MotB Complex of Escherichia coli

机译：大肠杆菌MotA / MotB复合物的增溶和纯化

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摘要

Bacterial flagella are driven at their base by a rotary motor fueled by the membrane gradient of protons or sodium ions.The stator of the flagellar motor is formed from the membrane proteins MotA and MotB,which function together to conduct ions across the membrane and couple ion flow to rotation.An invariant aspartate residue in MotB (Asp32 in the protein of E.coli) is essential for rotation and appears to have a direct role in proton conduction.A recent study showed that changes at Asp32 in MotB cause a conformational change in the complex,as evidenced by altered patterns of protease susceptibility of MotA [Kojima,S.,and Blair,D.F.(2001) Biochemistry 40 (43),13041-13050].It was proposed that protonation/deprotonation of Asp32 might regulate a conformational change in the stator that acts as the powerstroke to drive rotation of the rotor.Biochemical studies of the MotA/MotB complex have been hamepred by the absence of a suitable assay for its integrity in detergent solution.Here,we have studied the behavior of the MotA/MotB complex in a variety of detergents,making use of the protease-susceptibility assay to monitor its integrity.Among about 25 detergents tested,a few were found to solubilize the proteins effectively while preserving certain conformational properties characteristic of an intact complex.The detergent dodecylphosphocholine,or DPC,proved especially effective.MotA/MotB complexes purified in DPC migrate with an apparent size of approx300 kDa in gel-filtration columns,and retain the Asp32-modulated conformational differences seen in membranes.~(35)S-radiolabeling showed that MotA and MotB are present in a 2:1 ratio in the complex.Purified MotA/MotB complexes should enable in vitro study of the proton-induced conformational change and other aspects of stator function.
机译：细菌鞭毛的基部由质子或钠离子的膜梯度驱动的旋转电机驱动。鞭毛电机的定子由膜蛋白MotA和MotB形成，它们共同起着跨膜传导离子和耦合离子的作用。 MotB中恒定的天冬氨酸残基（大肠杆菌蛋白中的Asp32）对于旋转至关重要，并且似乎在质子传导中具有直接作用。最近的一项研究表明MotB中Asp32的变化会引起分子构象变化。由MotA的蛋白酶敏感性模式改变所证实的复合物[Kojima，S。，and Blair，DF（2001）Biochemistry 40（43），13041-13050]。有人提出，Asp32的质子化/去质子化可能调节构象。 MotA / MotB复合物的生化研究因缺乏在去污剂溶液中完整性的合适测定方法而受到阻碍，因此阻碍了MotA / MotB复合物的生化研究。 e。通过蛋白酶敏感性试验来监测MotA / MotB复合物在各种去污剂中的行为，以监测其完整性。在测试的25种去污剂中，发现有几种能有效溶解蛋白质，同时保留某些构象特性经证实，去污剂十二烷基磷酸胆碱或DPC尤其有效。在DPC中纯化的MotA / MotB复合物在凝胶过滤柱中迁移时的表观大小约为300 kDa，并保留了膜中可见的Asp32调节的构象差异。 （35）S-放射标记显示复合物中MotA和MotB的比例为2：1。纯化的MotA / MotB复合物应能在体外研究质子诱导的构象变化和定子功能的其他方面。

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