Wu J, Hardy D, Kaback H R
Department of Physiology, Howard Hughes Medical Institute, Molecular Biology Institute, University of California, Los Angeles 90095-1662, USA.
Biochemistry. 1999 Feb 9;38(6):1715-20. doi: 10.1021/bi982342b.
The N-terminal six transmenbrane helices (N6) and the C-terminal six transmembrane helices (C6) of the lactose permease, each containing a single-Cys residue, were coexpressed, and proximity was studied. Paired Cys residues in helices IV (positions 114, 116, 119, 122, 125, or 129) and VII (227, 231, 232, 234, 235, 238, 239, 242, 243, 245, or 246) or XI (350, 353, 354, 357, 361, or 364) were tested for cross-linking in the presence of two rigid homobifunctional thiol-specific cross-linkers, N,N'-o-phenylenedimaleimide (o-PDM; 6 A) and N,N'-p-phenylenedimaleimide (p-PDM; 10 A). Cys residues in the middle of helix IV (position 119 or 122) cross-link to Cys residues in the middle of helix VII (position 238, 239, 242, or 243). In contrast, no cross-linking is evident with paired Cys residues at either end of helix IV (position 114, 116, 125, or 129) or helix VII (position 227, 231, 232, 234, 235, 245, or 246). On the other hand, Cys residues in the cytoplasmic half of helix IV (position 125 or 129) cross-link with Cys residues in the cytoplasmic half of helix XI (position 350, 353, or 354), while paired Cys residues at the periplasmic ends of the two helices do not cross-link. The results indicate that helices IV and VII cross in a scissors-like manner with the cytoplasmic end of helix IV tilting toward helix XI.
乳糖通透酶的N端六个跨膜螺旋（N6）和C端六个跨膜螺旋（C6），每个都含有一个单半胱氨酸残基，将它们共表达并研究其接近程度。对螺旋IV（第114、116、119、122、125或129位）和螺旋VII（第227、231、232、234、235、238、239、242、243、245或246位）以及螺旋XI（第350、353、354、357、361或364位）中的成对半胱氨酸残基，在两种刚性同型双功能硫醇特异性交联剂N，N'-邻苯二甲酰亚胺马来酰亚胺（o-PDM；6 Å）和N，N'-对苯二甲酰亚胺马来酰亚胺（p-PDM；10 Å）存在的情况下进行交联测试。螺旋IV中间的半胱氨酸残基（第119或122位）与螺旋VII中间的半胱氨酸残基（第238、239、242或243位）发生交联。相反，螺旋IV两端（第114、116、125或129位）或螺旋VII两端（第227、231、232、234、235、245或246位）的成对半胱氨酸残基没有明显的交联。另一方面，螺旋IV胞质侧的半胱氨酸残基（第125或129位）与螺旋XI胞质侧的半胱氨酸残基（第350、353或354位）发生交联，而两个螺旋周质端的成对半胱氨酸残基不发生交联。结果表明，螺旋IV和VII以剪刀状方式交叉，螺旋IV的胞质端向螺旋XI倾斜。
