Targeted disulfide cross-linking of the MotB protein of Escherichia coli:: Evidence for two H+ channels in the stator complex
Targeted disulfide cross-linking of the MotB protein of Escherichia coli:: Evidence for two H+ channels in the stator complex
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DOI：
10.1021/bi011264g
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发表时间：
2001-10-30
期刊：
影响因子：
2.9
通讯作者：
Blair, DF
中科院分区：
文献类型：
--
作者：
Braun, TF;Blair, DF
细菌鞭毛由旋转马达转动，这些马达从质子或钠离子的膜梯度中获得能量。鞭毛马达的定子是由膜蛋白MotA和MotB组成的，它们结合在包含每种蛋白的多个拷贝的复合体中。这些配合物通过膜传导离子，并通过一种似乎涉及构象变化的机制将离子偶联流到马达旋转中[Kojima， S., and Blair， D. F. (2001) Biochemistry 40, 13041-13050]。关于MotA/MotB复合物的结构信息非常有限。MotA有四个跨膜节段，而MotB只有一个。我们已经开始了一项有针对性的二硫交联研究，以探索MotA/MotB复合物中膜段的排列，从MotB的单个膜段开始。在片段中连续21个位置引入了Cys残基，并在膜或洗涤剂溶液中研究了MotA/MotB配合物的二硫交联。大多数cys取代的MotB蛋白在氧化后形成二硫化物连接的二聚体，产量显著。二聚体的产率随Cys取代的位置有规律地变化，遵循平行对称的a-螺旋二聚体的模式。在基于交联实验的结构模型中，被认为促进质子运动的关键Asp32残基位于MotB二聚体的不同表面，因此可能在两个不同的质子通道中起作用。通过测量引入的半胱氨酸残基对n -乙基马来酰亚胺和带电荷的甲乙硫代磺酸试剂的反应活性，绘制了溶剂可接近的区域。靠近中间部分的位置是巯基试剂无法进入的。任何一端6-8个残基内的位置，包括Asp32周围的残基，都是可访问的。
Bacterial flagella are turned by rotary motors that obtain energy from the membrane gradient of protons or sodium ions. The stator of the flagellar motor is formed from the membrane proteins MotA and MotB, which associate in complexes that contain multiple copies of each protein. The complexes conduct ions across the membrane, and couple ion flow to motor rotation by a mechanism that appears to involve conformational changes [Kojima, S., and Blair, D. F. (2001) Biochemistry 40, 13041-13050]. Structural information on the MotA/MotB complex is very limited. MotA has four membrane-spanning segments, and MotB has one. We have begun a targeted disulfide-cross-linking study to probe the arrangement of membrane segments in the MotA/MotB complex, beginning with the single membrane segment of MotB. Cys residues were introduced in 21 consecutive positions in the segment, and disulfide cross-linking was studied in MotA/MotB complexes either in membranes or detergent solution. Most of the Cys-substituted MotB proteins formed disulfide-linked dimers in significant yield upon oxidation. The yield of dimer varied regularly with the position of the Cys substitution, following the pattern expected for a parallel, symmetric dimer of a-helices. In a structural model based on the cross-linking experiments, critical Asp32 residues that are believed to facilitate proton movement are positioned on separate surfaces of the MotB dimer and so probably function within two distinct proton channels. Regions accessible to solvent were mapped by measuring the reactivity of introduced Cys residues toward N-ethyl maleimide and a charged methanethiosulfonate reagent. Positions near the middle of the segment were inaccessible to sulhydryl reagents. Positions within 6-8 residues of either end, which includes residues around Asp32, were accessible.