Wang Q, Kaback H R
Howard Hughes Medical Institute, Department of Physiology, Molecular Biology Institute, University of California at Los Angeles 90095-1662, USA.
Biochemistry. 1999 Dec 21;38(51):16777-82. doi: 10.1021/bi991853a.
The six N-terminal transmembrane helices (N(6)) and the six C-terminal transmembrane helices (C(6)) in the lactose permease of Escherichia coli, each containing a single Cys residue, were coexpressed, and cross-linking was studied. The proximity of paired Cys residues in helices III (position 78, 81, 84, 86, 87, 88, 90, 93, or 96) and VII (position 227, 228, 231, 232, 235, 238, 239, 241, 243, 245, or 246) was examined by using iodine or two rigid homobifunctional thiol-specific cross-linking reagents with different lengths [N,N'-o-phenylenedimaleimide (o-PDM; 6 A) and N, N'-p-phenylenedimaleimide (p-PDM; 10 A)]. Cys residues in the periplasmic half of helix III (position 87, 93, or 96) cross-link to Cys residues in the periplasmic half of helix VII (position 235, 238, 239, 241, or 245). In contrast, no cross-linking is evident with paired Cys residues near the cytoplasmic ends of helices III (position 78 or 81) and VII (position 227, 228, 213, 232, or 235). Therefore, the periplasmic halves of helices III and VII are in close proximity, and the helices tilt away from each other toward the cytoplasmic face of the membrane. On the basis of the findings, a modified helix packing model for the permease is presented.
大肠杆菌乳糖通透酶中的6个N端跨膜螺旋（N(6)）和6个C端跨膜螺旋（C(6)），每个都含有一个半胱氨酸残基，将它们共表达并研究交联情况。通过使用碘或两种不同长度的刚性同型双功能硫醇特异性交联试剂[N,N'-邻苯二甲酰亚胺（o-PDM；6 Å）和N,N'-对苯二甲酰亚胺（p-PDM；10 Å）]，检测螺旋III（位置78、81、84、86、87、88、90、93或96）和螺旋VII（位置227、228、231、232、235、238、239、241、243、245或246）中配对半胱氨酸残基的接近程度。螺旋III周质侧的半胱氨酸残基（位置87、93或96）与螺旋VII周质侧的半胱氨酸残基（位置235、238、239、241或245）发生交联。相反，螺旋III（位置78或81）和螺旋VII（位置227、228、213、232或235）胞质端附近的配对半胱氨酸残基没有明显的交联。因此，螺旋III和螺旋VII的周质侧相互靠近，并且螺旋朝着膜的胞质面彼此倾斜。基于这些发现，提出了一种通透酶的改良螺旋堆积模型。
