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首页> 外文期刊>Biochemistry >Barriers to Folding of the Transmembrane Domain of the Escherichia coli Autotransporter Adhesin Involved in Diffuse Adherence
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Barriers to Folding of the Transmembrane Domain of the Escherichia coli Autotransporter Adhesin Involved in Diffuse Adherence

机译：折叠大肠杆菌的跨膜结构域的障碍涉及弥漫性粘附的粘附粘剂

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摘要

Adhesin involved in diffuse adherence(AIDA)is an autotransporter protein that confers the diffuse adherence phenotype to certain diarrheagenic Escherichia coli strains.It consists of a 49 amino acid signal peptide,a 797 amino acid passenger domain,and a 440 amino acid beta-domain integrated in the outer membrane.The beta-domain consists of two parts:the betai-domain,which is predicted to form two beta-strands on the bacterial cell surface,and the beta_2-domain,which constitutes the transmembrane domain.We here present a detailed biophysical analysis of the AIDA beta-domain addressing its refolding properties and its different conformational states and their stability.We find that the beta_2-domain in solution can fold only when the beta_1-domain is present and only with 50% efficiency.However,100% refolding of the beta_2-domain,with or without the beta_1-domain,can be achieved in the presence of a solid support.Folding can only take place above the cmc of the detergent used,but the refolded state is retained if diluted below the cmc,revealing a kinetic barrier to dissociation of the detergent molecules from the folded protein.Refolding attempts of the beta_2-domain in the absence of a solid support result in the formation of an oligomeric misfolded state both in the absence and in the presence of detergent.Despite being misfolded,these states unfold cooperatively with a T_m approx= 70 deg C.The refolded protein in the nonionic detergent octylpolyoxyethylene(oPOE)can only be thermally unfolded in the presence of SDS.The linear relationship between SDS mole fraction and unfolding temperature,T_m,predicts a T_m of 112.9(+-)1.2 deg C for the beta_2-domain and 132.7(+-)12.2 deg C for the entire beta-domain in pure oPOE.Thus,the betai-domain also stabilizes the beta_2-domain.In conclusion,our data show that the in vitro refolding of the AIDA beta-domain is critically dependent on a solid support,suggesting that in vivo specific biological factors may assist in folding the protein correctly into the outer membrane to avoid the formation of stably misfolded conformations.
机译：涉及弥漫性粘附（AIDA）的粘附蛋白是一种自耦体运动蛋白，其将漫反射粘附表型赋予某些腹泻的大肠杆菌菌株。由49个氨基酸信号肽，797个氨基酸乘客区和440个氨基酸β-结构域组成。集成在外膜中。β-结构域由两部分组成：BetaI结构域，预计在细菌细胞表面上形成两个β-股，并且β-2结构域构成跨膜结构域。这里对其重折叠特性及其不同构象状态的AIDAβ域的详细生物物理分析及其稳定性。我们发现溶液中的Beta_2-域只能折叠β1-域，只有50％的效率。然而，在固体载体的存在下，可以在存在的存在下实现100％的β_2-结构域的重折叠，有或没有β_1-结构域。倍数只能在所用洗涤剂的CMC上方发生，但是如果在CMC下方稀释，则保留旧状态，揭示来自折叠蛋白的洗涤剂分子的动力学阻隔。在没有固体支持的情况下，在不存在固体载体的情况下，β2-结构域的尝试将形成寡聚粘附状态缺席和存在的洗涤剂存在。分解的缺失，这些状态与T_M约= 70℃展开。非离子洗涤剂辛基聚乙烯（OPOE）中的重折叠蛋白只能在SDS存在下热膨胀。线性SDS摩尔分数与展开温度，T_M之间的关系，预测β-2域112.9（+ - ）1.2℃的T_m，132.7（+ - ）12.2℃下为纯opoe.thus， Betai-Domain还稳定了β-2-domain.in结论，我们的数据表明，AIDAβ结构域的体外重叠均依赖于固体支持，表明在体内特定的生物因素中可以有助于折叠折叠蛋白质正确地进入外膜以避免形成稳定错误的折叠构象。

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  • 来源
    《Biochemistry》 |2005年第11期|共13页
  • 作者

    Jesper E.Mogensen; Damini Tapadar; M.Alexander Schmidt; Daniel E.Otzen;

  • 作者单位

    Department of Life Sciences Aalborg University Sohngaardsholmsvej 49 DK-9000 Aalborg Denmark and Institut fur Infektiologie Zentrum fur Molekularbiologie der Entzundung(ZMBE) Westfalische Wilhelms-Universitat Munster Von-Esmarch-Strasse 56 D-48J49 Mu;

    Department of Life Sciences Aalborg University Sohngaardsholmsvej 49 DK-9000 Aalborg Denmark and Institut fur Infektiologie Zentrum fur Molekularbiologie der Entzundung(ZMBE) Westfalische Wilhelms-Universitat Munster Von-Esmarch-Strasse 56 D-48J49 Mu;

    Department of Life Sciences Aalborg University Sohngaardsholmsvej 49 DK-9000 Aalborg Denmark and Institut fur Infektiologie Zentrum fur Molekularbiologie der Entzundung(ZMBE) Westfalische Wilhelms-Universitat Munster Von-Esmarch-Strasse 56 D-48J49 Mu;

    Department of Life Sciences Aalborg University Sohngaardsholmsvej 49 DK-9000 Aalborg Denmark and Institut fur Infektiologie Zentrum fur Molekularbiologie der Entzundung(ZMBE) Westfalische Wilhelms-Universitat Munster Von-Esmarch-Strasse 56 D-48J49 Mu;

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