Braun T F, Blair D F
Department of Biology, University of Utah, Salt Lake City, Utah 84112, USA.
Biochemistry. 2001 Oct 30;40(43):13051-9. doi: 10.1021/bi011264g.
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 alpha-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.
细菌鞭毛由旋转马达驱动，该马达从质子或钠离子的膜梯度中获取能量。鞭毛马达的定子由膜蛋白MotA和MotB形成，它们以包含每种蛋白质多个拷贝的复合物形式结合。这些复合物介导离子跨膜运输，并通过一种似乎涉及构象变化的机制将离子流动与马达旋转耦合起来[小岛，S.，和布莱尔，D. F.（2001年）《生物化学》40卷，13041 - 13050页]。关于MotA/MotB复合物的结构信息非常有限。MotA有四个跨膜片段，MotB有一个。我们已经开始了一项有针对性的二硫键交联研究，以探究MotA/MotB复合物中膜片段的排列方式，从MotB的单个膜片段开始。在该片段的21个连续位置引入了半胱氨酸残基，并在膜或去污剂溶液中的MotA/MotB复合物中研究了二硫键交联。大多数半胱氨酸取代的MotB蛋白在氧化后以显著产率形成二硫键连接的二聚体。二聚体的产率随半胱氨酸取代位置的变化而有规律地变化，遵循α - 螺旋平行对称二聚体预期的模式。在基于交联实验的结构模型中，被认为有助于质子移动的关键天冬氨酸32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内发挥作用。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硫基磺酸盐试剂的反应性，绘制了可及溶剂区域。该片段中间附近的位置对巯基试剂不可及。两端6 - 8个残基范围内的位置，包括天冬氨酸32周围的残基，是可及的。
