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首页> 外文期刊>Biochemistry >Site-directed sulfhydryl labeling of the lactose permease of Escherichia coli: helix X.
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Site-directed sulfhydryl labeling of the lactose permease of Escherichia coli: helix X.

机译：大肠杆菌乳糖通透酶的定点巯基标记：螺旋X。

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摘要

Helix X in the lactose permease of Escherichia coli contains two residues that are irreplaceable with respect to active transport, His322 and Glu325, as well as Lys319, which is charge-paired with Asp240 in helix VII. Structural and dynamic features of transmembrane helix X are investigated here by site-directed thiol modification of 14 single-Cys replacement mutants with N-[(14)C]ethylmaleimide (NEM) in right-side-out membrane vesicles. Permease mutants with a Cys residue at position 326, 327, 329, 330, or 331 in the cytoplasmic half of the transmembrane domain are alkylated by NEM at 25 degrees C, a mutant with Cys at position 315 at the periplasmic surface is labeled in the presence of substrate exclusively, and mutants with Cys at positions 317, 318, 320, 321, 324, 328, 332, or 333 do not react with NEM under the conditions tested. Binding of substrate causes increased labeling of a Cys residue at position 315 and decreased labeling of Cys residues at positions 326, 327, and 329. Studies with methanethiosulfonate ethylsulfonate indicate that Cys residues at positions 326, 329, 330, and 331 in the cytoplasmic half are accessible to the aqueous phase from the periplasmic face of the membrane. Ligand binding results in clear attenuation of solvent accessibility of Cys at position 326 and a marginal increase in accessibility of Cys at position 327 to solvent. The findings indicate that the cytoplasmic half of helix X is more reactive/accessible to thiol reagents and more exposed to solvent than the periplasmic half. Furthermore, positions that reflect ligand-induced conformational changes are located on the same face of helix X as Lys319, His322, and Glu325.
机译：大肠杆菌的乳糖通透酶中的螺旋X包含两个相对于活性转运不可替代的残基：His322和Glu325，以及Lys319，后者与螺旋VII中的Asp240电荷配对。跨膜螺旋X的结构和动态特征在这里通过对N-[（14）C]乙基马来酰亚胺（NEM）的14个单Cys置换突变体进行定点巯基修饰，在右侧向外的膜囊泡中进行研究。在25°C下通过NEM将跨膜结构域的胞质半部分中326、327、329、330或331位Cys残基的通透酶突变体烷基化，在周质表面标记315位Cys的突变体。在测试条件下，仅存在底物，以及在317、318、320、321、324、328、332或333位具有Cys的突变体不会与NEM反应。底物的结合导致在315位的Cys残基的标记增加，而在326、327和329位的Cys残基的标记减少。甲硫基磺酸乙酯的研究表明，胞质半部中326、329、330和331位的Cys残基从膜的周质面可接近水相。配体结合导致位置326上Cys的溶剂可及性明显减弱，并且位置327上Cys的溶剂可及性略有增加。研究结果表明，螺旋X的胞质半部比周质半部对硫醇试剂更具反应性/可及性，并且更多地暴露于溶剂中。此外，反映配体诱导的构象变化的位置与Lys319，His322和Glu325位于螺旋X的同一面上。

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