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首页> 外文期刊>Journal of bacteriology >Permissive sites and topology of an outer membrane protein with a reporter epitope.
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Permissive sites and topology of an outer membrane protein with a reporter epitope.

机译：带有报道分子表位的外膜蛋白的允许位点和拓扑。

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We are developing a genetic approach to study with a single antibody the folding and topology of LamB, an integral outer membrane protein from Escherichia coli K-12. This approach consists of inserting the same reporter foreign antigenic determinant (the C3 epitope from poliovirus) at different sites of LamB so that the resulting hybrid proteins have essentially kept the in vivo biological properties of LamB and therefore its cellular location and structure; the corresponding sites are called permissive sites. A specific monoclonal antibody can then be used to examine the position of the reporter epitope with respect to the protein and the membrane. We present an improved and efficient procedure that led us to identify eight new permissive sites in LamB. These sites appear to be distributed on both sides of the membrane. At one of them (after residue 253), the C3 epitope was detected on intact bacteria, providing the first direct argument for exposure of the corresponding LamB region at the cell surface. At this site as well as at four others (after residues 183, 219, 236, and 352), the C3 epitope could be detected with the C3 monoclonal antibody at the surface of the extracted trimeric LamB-C3 hybrid proteins. We provide a number of convergent arguments showing that the hybrid proteins are not strongly distorted with respect to the wild-type protein so that the conclusions drawn are also valid for this protein. These conclusions are essentially in agreement with the proposed folding model for the LamB protein. They agree, in particular, with the idea that regions 183 and 352 are exposed to the periplasm. In addition, they suggest that region 236 is buried at the external face of the outer membrane and that region 219 is exposed to the periplasm. Including the 3 sites previously determined, 11 permissive sites are now available in LamB, including 3 at the cell surface and most probably at least 3 in the periplasm. We discuss the nature of such sites, the generalization of this approach to other proteins, and possible applications.
机译：我们正在开发一种遗传学方法，用一种抗体研究LamB（大肠杆菌K-12中不可或缺的外膜蛋白）的折叠和拓扑。这种方法包括在LamB的不同位点插入相同的报告基因外源抗原决定簇（脊髓灰质炎病毒的C3表位），从而使所得的杂合蛋白基本保持LamB的体内生物学特性，从而保持其细胞位置和结构。相应的站点称为许可站点。然后可以使用特定的单克隆抗体来检查报告基因表位相对于蛋白质和膜的位置。我们提出了一种改进的高效程序，使我们在LamB中确定了八个新的许可站点。这些位点似乎分布在膜的两侧。在其中一个中（在残基253之后），在完整细菌上检测到C3表位，这是在细胞表面暴露相应的LamB区的第一个直接依据。在此位点以及其他四个位点（在残基183、219、236和352之后），可以使用C3单克隆抗体在提取的三聚LamB-C3杂合蛋白表面检测C3表位。我们提供了许多收敛的论据，表明杂合蛋白相对于野生型蛋白没有强烈变形，因此得出的结论对该蛋白也有效。这些结论与拟议的LamB蛋白折叠模型基本吻合。他们尤其同意区域183和352暴露于周质的想法。另外，他们建议区域236被掩埋在外膜的外表面，并且区域219暴露于周质。包括先前确定的3个位点，现在LamB中有11个允许的位点，其中3个位于细胞表面，很可能至少3个位于周质中。我们讨论了此类位点的性质，这种方法对其他蛋白质的推广以及可能的应用。

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