Helix packing of lactose permease in Escherichia coli studied by site-directed chemical cleavage.
Helix packing of lactose permease in Escherichia coli studied by site-directed chemical cleavage.
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通过定点化学裂解研究大肠杆菌中乳糖通透酶的螺旋包装。
DOI：
10.1073/pnas.92.20.9186
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发表时间：
1995
影响因子：
11.1
通讯作者：
Kaback Hr
中科院分区：
文献类型：
--
作者：
Jianhua Wu;David M. Perrin;D. Sigman;Kaback Hr
从大肠杆菌中提取的生物素化乳糖渗透酶含有330位（螺旋X）或147、148或149位（螺旋V）的单半胱氨酸残基，通过亲和层析纯化，并用5-（α -溴乙酰氨基）-1,10-菲罗啉铜[OP(Cu)]衍生化。对纯化的OP（Cu）标记的十二烷基- β - d -麦芽吡苷中Leu-330—b>胱氨酸渗透酶的研究表明，在抗坏血酸存在下孵育后，在抗c端抗体的免疫印迹上观察到约19和6-8 kDa的裂解产物。值得注意的是，当渗透酶嵌入天然膜时，观察到相同的裂解产物。与独立表达的通透酶的c端一半作为标准进行比较表明，19 kda的产物是由螺旋VII细胞质端附近的分裂产生的，而6-至8 kda的片段可能是由螺旋XI细胞质端附近的分裂产生的。结果与早期的位点定向荧光和位点定向诱变研究得出的渗透酶c端一半的三级结构模型完全一致。对OP（Cu）标记的Cys-148渗透酶的类似研究显示，裂解产物约为19 kDa和15-16 kDa。较大的片段可能反映了螺旋VII细胞质端附近的切割，而15至16 kda的片段则与螺旋VIII细胞质端附近的切割一致。当OP（Cu）移动100度到位置149 （Val-149—> Cys渗透酶）时，在19 kDa处观察到一个单一的产物，表明螺旋VII的细胞质端断裂。然而，当试剂在另一个方向移动100度到位置147 （Gly-147—> Cys渗透酶）时，没有观察到裂解。结果表明，螺旋V靠近螺旋VII和螺旋VIII， 148位位于螺旋界面，149位面向螺旋VII， 147位面向脂质双分子层。
Biotinylated lactose permease from Escherichia coli containing a single-cysteine residue at position 330 (helix X) or at position 147, 148, or 149 (helix V) was purified by avidin-affinity chromatography and derivatized with 5-(alpha-bromoacetamido)-1,10-phenanthroline-copper [OP(Cu)]. Studies with purified, OP(Cu)-labeled Leu-330 --> Cys permease in dodecyl-beta-D-maltopyranoside demonstrate that after incubation in the presence of ascorbate, cleavage products of approximately 19 and 6-8 kDa are observed on immunoblots with anti-C-terminal antibody. Remarkably, the same cleavage products are observed with permease embedded in the native membrane. Comparison with the C-terminal half of the permease expressed independently as a standard indicates that the 19-kDa product results from cleavage near the cytoplasmic end of helix VII, whereas the 6- to 8-kDa fragment probably results from fragmentation near the cytoplasmic end of helix XI. Results are entirely consistent with a tertiary-structure model of the C-terminal half of the permease derived from earlier site-directed fluorescence and site-directed mutagenesis studies. Similar studies with OP(Cu)-labeled Cys-148 permease exhibit cleavage products at approximately 19 kDa and at 15-16 kDa. The larger fragment probably reflects cleavage at a site near the cytoplasmic end of helix VII, whereas the 15- to 16-kDa fragment is consistent with cleavage near the cytoplasmic end of helix VIII. When OP(Cu) is moved 100 degrees to position 149 (Val-149 --> Cys permease), a single product is observed at 19 kDa, suggesting fragmentation at the cytoplasmic end of helix VII. However, when the reagent is moved 100 degrees in the other direction to position 147 (Gly-147 --> Cys permease), cleavage is not observed. The results suggest that helix V is in close proximity to helices VII and VIII with position 148 in the interface between the helices, position 149 facing helix VII, and position 147 facing the lipid bilayer.