Department of Physiology, University of California, Los Angeles, California 90095, United States.
Biochemistry. 2011 Mar 15;50(10):1634-40. doi: 10.1021/bi101988s. Epub 2011 Feb 8.
In total, 59 single Cys-replacement mutants in helix VII and helix X of the lactose permease of Escherichia coli were subjected to site-directed fluorescence labeling in right-side-out membrane vesicles to complete the testing of Cys accessibility or reactivity. For both helices, accessibility/reactivity is relatively low at the level of the sugar-binding site where the helices are tightly packed. However, labeling of Cys substitutions in helix VII with tetramethylrhodamine-5-maleimide decreases from the middle toward the cytoplasmic end and increases toward the periplasmic end. Helix X is labeled mainly on the side facing the central hydrophilic cavity with relatively small or no changes in the presence of ligand. In contrast, sugar binding causes a significant increase in accessibility/reactivity at the periplasmic end of helix VII. When considered with similar findings from N-ethylmaleimide alkylation studies, the results confirm and extend support for the alternating access model.
共有 59 种单半胱氨酸替换突变体被用于大肠杆菌乳糖通透酶的 VII 螺旋和 X 螺旋的定点荧光标记，以完成对半胱氨酸可及性或反应性的测试。对于这两个螺旋，在糖结合位点水平上，螺旋紧密堆积，可及性/反应性相对较低。然而，用四甲基罗丹明-5-马来酰亚胺标记 VII 螺旋中的半胱氨酸取代物，从中部朝向细胞质末端逐渐减少，而朝向周质末端逐渐增加。X 螺旋主要在面向中央亲水区的一侧被标记，在有配体存在的情况下，变化相对较小或没有变化。相比之下，糖结合会导致 VII 螺旋的周质末端的可及性/反应性显著增加。当与 N-乙基马来酰亚胺烷基化研究的类似发现结合考虑时，这些结果证实并扩展了对交替访问模型的支持。
