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首页> 外文期刊>Biochemistry >Cysteine-Scanning Mutagenesis and Thiol Modification of the Rickettsia prowazekii ATP/ADP Translocase:Evidence That Transmembrane Regions I and II,but Not III,Are Structural Components of the Aqueous Translocation Channel
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Cysteine-Scanning Mutagenesis and Thiol Modification of the Rickettsia prowazekii ATP/ADP Translocase:Evidence That Transmembrane Regions I and II,but Not III,Are Structural Components of the Aqueous Translocation Channel

机译：立克次体ATP / ADP转移酶的半胱氨酸扫描诱变和硫醇修饰：跨膜区域I和II，但不是III，是水易位通道的结构成分的证据。

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摘要

The contribution of transmembrane regions I,II,and III of the Rickettsia prowazekii ATP/ ADP translocase to the structure of the putative water-filled ATP translocation channel was evaluated from the accessibility of hydrophilic,thiol-reactive,methanethiosulfonate reagents to a library of 68 independent cysteine-substitution mutants heterologously expressed in Escherichia coli.The MTS reagents used were MTSES (negatively charged) and MTSET and MTSEA (both positively charged).Mutants F036C,Y042C,and R046C (TM I),K066C and P072C (TM II),and F101C,F105C,F108C,Y113C,and P114C (TM III) had no assay able transport activity,indicating that cysteine substitution at these positions may not be tolerated.All three MTS reagents inhibit the transport of ATP in mutants of TM I (L039C,S043C,S047C,I048C) and TM II (S061C,S063C,T067C,I069C,V070C,A074C).Further,these residues appear to cluster along a single face of the transmembrane domain.Preexposure of MTS-reactive mutants S047C (TM I) and T067C (TM II) to high levels of ATP resulted in protection from MTS-mediated inhibition.This indicated that both TM I and TM II make major contributions to the structure of an aqueous ATP translocation pathway.Finally,on the basis of the lack of accessibility of charged MTS reagents to the thiol groups in mutants of TM III,it appears that TM III is not exposed to the ATP translocation channel.Cysteine substitution of residues constituting a highly conserved "phenylalanine face" in TM III resulted in ablation of ATP transport activity.Further,substituting these phenylalanine residues for either isoleucine or tyrosine also resulted in much lower transport activity,indicating that some property of phenylalanine at these positions that is not shared by cysteine,isoleucine,or tyrosine is critical to translocase activity.
机译：从亲水的，可与硫醇反应的甲硫代磺酸盐试剂进入68个文库的可及性评估了立克次氏体立克次体ATP / ADP跨膜酶的跨膜区域I，II和III对假定的充满水的ATP易位通道结构的贡献在大肠杆菌中异源表达的独立半胱氨酸替代突变体。使用的MTS试剂为MTSES（带负电），MTSET和MTSEA（均为带正电）。突变体F036C，Y042C和R046C（TM I），K066C和P072C（TM II） ，F101C，F105C，F108C，Y113C和P114C（TM III）没有可检测的转运活性，表明这些位置上的半胱氨酸取代可能不被接受。所有三种MTS试剂均抑制TM I突变体中ATP的转运（ L039C，S043C，S047C，I048C）和TM II（S061C，S063C，T067C，I069C，V070C，A074C）。此外，这些残基似乎沿跨膜结构域的单个表面聚集.MTS反应性突变体S047C（TM）的预暴露I）和T067C（TM II）高水平的ATP导致不受MTS介导的抑制作用的保护。这表明TM I和TM II均对水性ATP易位途径的结构做出了主要贡献。最后，在缺乏电荷的可及性的基础上用MTS试剂分析TM III突变体中的硫醇基，看来TM III没有暴露于ATP易位通道。半胱氨酸取代了TM III中构成高度保守的“苯丙氨酸面”的残基，从而消除了ATP转运活性。用苯丙氨酸残基取代异亮氨酸或酪氨酸也会导致转运活性低得多，这表明半胱氨酸，异亮氨酸或酪氨酸不具有的这些位置上苯丙氨酸的某些特性对转移酶活性至关重要。

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