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首页> 外文期刊>Biochemistry >Proximity of Cytoplasmic and Periplasmic Loops in NhaA Na~+/H~+ Antiporter of Escherichia coli As Determined by Site-Directed Thiol Cross-Linking
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Proximity of Cytoplasmic and Periplasmic Loops in NhaA Na~+/H~+ Antiporter of Escherichia coli As Determined by Site-Directed Thiol Cross-Linking

机译：由位点导向硫醇交联测定的大肠杆菌的NHAA Na〜+ / H〜+ + + + + + + + + + + + + +丙二醇的接近

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摘要

The unique trypsin cleavage site of NhaA, the Na~+/H~+ antiporter of Escherichia coli, was exploited to detect a change in mobilit of cross-linked products of NhaA by polyacrylamide gel electrophoresis. Double-Cys replacements were introduced into loops, one on each side of the trypsin clevage site (Lys 249). The proximity of paired Cys residues was assessed by disfuldie cross-linking of the two tryptic fragments, using three homobifunctional cross-linking agents: 1,6-bis9maleimido)hexane (BMH), N,N'-o-phenylenedimaleimide (o-PDM), and N,N'-p-phenylenedimaleimide (p-PDM). The interloop cross-linking was found to be very specific, indicating that the loops are not merely random coils that interact randomy. In the periplasmic side of NhaA, two patterns of cross-linking are observed: (a) all three cross-linking reagents cross-link very efficiently between the double-Cys replacements A118C/S286C, N177C/S352C, and H255C/S352C; (b) only BMH cross-links the double-Cys replacements A118C/S352C N177C/S286C, and H225C/S286C. In the cytoplasmic side of NhaA, three patterns of cross-linking are observed: (a) all three cross-linking reagents cross-link very efficiently the pairs of Cys replacements L4C/E252C, S146C/L316C, S146C/R383C, and E241C/E252C; (b) BMH and p-PDM cross-link efficiently the pairs of Cys replacements S87C/E252C, S87C/L316C, and S146C/E252C (c) none of the reagents cross-links the double-Cys replacements L4C/L316C, L4C/R383C, S87C/R383C, A202C/E252C, A202C/L316C, A202C/R383C, E241C/L316C, and E241C/R383C. The data reveal that the N-terminus and loop VIII-IX that have previously been shown to change conformationn with pH are in close proximity within the NhaA protein. The data also suggest close proximity between N-terminal and C-terminal helicese at both the cytoplasmic and the periplasmic face of NhaA.
机译：利用聚丙烯酰胺凝胶电泳，利用了NHAA的NHAA，NA + / H〜+ +炔替替斯的NHAA的NAA〜+ / H〜+炔醇蛋白，通过聚丙烯酰胺凝胶电泳检测了NHAA交联产物的变化。将双Cys更换换成环，在胰蛋白酶夹层的每一侧（Lys 249）。通过三种胰蛋白酶片段的倾斜交联评估配对Cys残留物的接近，使用三种同型偶联的交联剂：1,6-双偶联的交联剂，N，N'-O-苯二维咪唑酰亚胺酰亚胺（O-PDM ），N，N'-P-苯二维咪唑（P-PDM）。发现界面交叉链接非常具体，表明环路不仅仅是互动的随机线圈。在NHAA的周质侧，观察到两种交联模式：（a）所有三种交联试剂在双Cys替换之间非常有效地交联，在双Cys替换A118C / S286C，N177C / S352C和H255C / S352C之间非常有效; （b）只有BMH交联双Cys替换A118C / S352C N177C / S286C和H225C / S286C。在NHAA的细胞质侧，观察到三种交联模式：（a）所有三个交联剂的交联试剂非常有效地通过替换L4C / E252C，S146C / L316C，S146C / R383C和E241C和E241C / E252C; （b）BMH和P-PDM交叉链路有效地替换S87C / E252C，S87C / L316C和S146C / E252C（C）无试剂交联双CYS替代L4C / L316C，L4C / R383C，S87C / R383C，A202C / E252C，A202C / L316C，A202C / R383C，E241C / L316C和E241C / R383C。数据显示，先前已被证明以pH值改变符合pH的N-末端和环VIII-IX在NHAA蛋白内密切接近。数据还表明N-Terminal和N-Terminal Helicess在NHAA的细胞质和周质面上的近距离接近。

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  • 来源
    《Biochemistry》 |2002年第50期|共9页
  • 作者

    A.Rimon; T.Tzubery; L.Galili; E.Padan;

  • 作者单位

    Alexander Silberman Institute of Life Sciences Hebrew University of Jerusalem 91904 Jerusalem Israel;

    Alexander Silberman Institute of Life Sciences Hebrew University of Jerusalem 91904 Jerusalem Israel;

    Alexander Silberman Institute of Life Sciences Hebrew University of Jerusalem 91904 Jerusalem Israel;

    Alexander Silberman Institute of Life Sciences Hebrew University of Jerusalem 91904 Jerusalem Israel;

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  • 正文语种 eng
  • 中图分类 生物化学;
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