Venkatesan P, Kwaw I, Hu Y, Kaback H R
Howard Hughes Medical Institute, Departments of Physiology and Microbiology & Molecular Genetics, Molecular Biology Institute, University of California-Los Angeles, Los Angeles, California 90095-1662, USA.
Biochemistry. 2000 Sep 5;39(35):10641-8. doi: 10.1021/bi000438b.
Site-directed sulfhydryl modification in situ is employed to investigate structural and dynamic features of transmembrane helix VII and the beginning of the periplasmic loop between helices VII and VIII (loop VII/VIII). Essentially all of the Cys-replacement mutants in the periplasmic half of the helix and the portion of loop VII/VIII tested are labeled by N-[(14)C]ethylmaleimide (NEM). In contrast, with the exception of two mutants at the cytoplasmic end of helix VII, none of the mutants in the cytoplasmic half react with the alkylating agent. Labeling of most of the mutants is unaltered by ligand at 25 degrees C. However, at 4 degrees C, conformational changes induced by substrate binding become apparent. In the presence of ligand, permease mutants with a Cys residue at position 241, 242, 244, 245, 246, or 248 undergo a marked increase in labeling, while the reactivity of a Cys at position 238 is slightly decreased. Labeling of the remaining Cys-replacement mutants is unaffected by ligand. Studies with methanethiosulfonate ethylsulfonate (MTSES), a hydrophilic impermeant thiol reagent, show that most of the positions that react with NEM are accessible to MTSES; however, the two NEM-reactive mutants at the cytoplasmic end of helix VII and position 236 in the middle of the membrane-spanning domain are not. The findings demonstrate that positions in helix VII that reflect ligand-induced conformational changes are located in the periplasmic half and accessible to the aqueous phase from the periplasmic face of the membrane. In the following papers in this issue (Venkatesan, P., Lui, Z., Hu, Y., and Kaback H. R.; Venkatesan, P., Hu, Y., and Kaback H. R.), the approach is applied to helices II and X.
采用原位定点巯基修饰法来研究跨膜螺旋VII以及螺旋VII和VIII之间周质环起始部分（环VII / VIII）的结构和动力学特征。基本上，螺旋周质半段和所测试的环VII / VIII部分中的所有半胱氨酸替代突变体都能被N - [（14）C]乙基马来酰亚胺（NEM）标记。相比之下，除了螺旋VII胞质端的两个突变体之外，胞质半段中的突变体均不与烷基化剂发生反应。在25℃时，大多数突变体的标记不受配体影响。然而，在4℃时，底物结合诱导的构象变化变得明显。在配体存在的情况下，在位置241、242、244、245、246或248处有半胱氨酸残基的通透酶突变体的标记显著增加，而位置238处半胱氨酸的反应性略有下降。其余半胱氨酸替代突变体的标记不受配体影响。用亲水性非渗透性硫醇试剂甲硫基磺酸乙酯（MTSES）进行的研究表明，大多数与NEM反应的位置可被MTSES接近；然而，螺旋VII胞质端的两个NEM反应性突变体以及跨膜结构域中间的位置236则不能。这些发现表明，反映配体诱导构象变化的螺旋VII中的位置位于周质半段，并且从膜的周质面可被水相接近。在本期后续的论文中（Venkatesan, P., Lui, Z., Hu, Y., and Kaback H. R.; Venkatesan, P., Hu, Y., and Kaback H. R.），该方法被应用于螺旋II和X。
