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首页> 外文学位 >Single deletion analysis and thiol cross -linking of a membrane transport protein: The lactose permease of Escherichia coli.
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Single deletion analysis and thiol cross -linking of a membrane transport protein: The lactose permease of Escherichia coli.

机译：膜转运蛋白的单缺失分析和巯基交联：大肠杆菌的乳糖通透酶。

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摘要

The lactose permease of Escherichia coli is an important model system for secondary active transport proteins. In lieu of a high resolution crystal structure, a battery of biochemical, biophysical, and genetic techniques have been developed in order to obtain both structural and functional information about this protein. This dissertation introduces single deletions as a method for approximating the termini of transmembrane domains of polytopic membrane proteins. The approach is based upon the postulate that alteration of the register of regular transmembrane structures by deletion of single amino acid residues will disrupt helix-helix interactions required for transport activity. In contrast, single deletion of loop residues should be relatively innocuous.;It is demonstrated that 19 of the 24 loop-helix interfaces approximated by loss or diminution of transport activity as the result of deletion of single amino acid residues are within 1 to 4 residues of the termini proposed by hydropathy analysis or other predictive algorithms. Three of the remaining five loop-helix interfaces are consistent with results from site-directed spin labeling studies but differ significantly from prediction. The results suggest that a strong correlation exists between structure and loss of activity as determined by single deletion analysis and support a topological model of the lactose permease consisting of 12 transmembrane alpha-helices with an average length of 21 residues.;A major change in topology suggested by deletion analysis and confirmed by site-directed spin labeling studies is the placement of the Glu126 and Arg144 into helices IV and V, respectively which are the major determinants for substrate binding. Utilizing site-directed thiol-crosslinking, it is shown that Cys residues in place of Glu126 and Arg144, as well as Ala122 and Val149 which are approximately one helical turn removed from the essential residues, spontaneously cross-link indicating that these residues are in close proximity. Neutralization of Glu126 or both Glu126 and Arg144 with Ala has little effect on the cross-linking of A122C/Val149C. In contrast, neutralization Arg144 with Ala results in marked decrease in cross-linking efficiency, indicating that an unpaired carboxylate at position 126 causes a structural perturbation at the interface between helices IV and V.
机译：大肠杆菌的乳糖通透酶是次级活性转运蛋白的重要模型系统。为了获得高分辨率的晶体结构，已经开发了一系列生物化学，生物物理和遗传技术，以便获得有关该蛋白质的结构和功能信息。本论文介绍了单缺失作为一种逼近多肽膜蛋白跨膜结构域末端的方法。该方法基于这样的假设，即通过缺失单个氨基酸残基来改变规则的跨膜结构的寄存器将破坏运输活性所需的螺旋-螺旋相互作用。相比之下，环残基的单个缺失应该是相对无害的；证明了24个环-螺旋界面中的19个（由于单个氨基酸残基的缺失而导致的运输活性的丧失或减少）近似在1-4个残基内亲水性分析或其他预测算法建议的末端。其余五个回路-螺旋界面中的三个与定点自旋标记研究的结果一致，但与预测有显着差异。结果表明，通过单缺失分析确定的结构与活性丧失之间存在很强的相关性，并支持了由12个跨膜α-螺旋组成的乳糖渗透酶的拓扑模型，平均长度为21个残基。通过缺失分析表明并经定点旋转标记研究证实的是将Glu126和Arg144分别置于螺旋IV和V中，这是底物结合的主要决定因素。利用定点硫醇交联技术，发现Cys残基取代了Glu126和Arg144以及Ala122和Val149，它们从基本残基中去除了大约一个螺旋角，自发地交联表明这些残基是紧密的接近。用Ala中和Glu126或Glu126和Arg144都不会对A122C / Val149C的交联产生影响。相反，用Ala中和Arg144会导致交联效率显着降低，这表明位置126处未配对的羧酸盐会在螺旋IV和V之间的界面处引起结构扰动。

著录项

  • 作者

    Wolin, Christopher Dean.;

  • 作者单位

    University of California, Los Angeles.;

  • 授予单位 University of California, Los Angeles.;
  • 学科 Microbiology.;Genetics.;Molecular biology.
  • 学位 Ph.D.
  • 年度 2001
  • 页码 59 p.
  • 总页数 59
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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