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首页> 外文期刊>Journal of Molecular Biology >The trimeric periplasmic chaperone skp of Escherichia coli forms 1 : 1 complexes with outer membrane proteins via hydrophobic and electrostatic interactions
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The trimeric periplasmic chaperone skp of Escherichia coli forms 1 : 1 complexes with outer membrane proteins via hydrophobic and electrostatic interactions

机译：大肠埃希氏菌的三聚体周质伴侣蛋白通过疏水和静电相互作用与外膜蛋白形成1：1复合物

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The interactions of outer membrane proteins (OMPs) with the periplasmic chaperone Skp from Escherichia coli are not well understood. We have examined the binding of Skp to various CIMPs of different origin, size, and function. These were OmpA, OmpG, and YaeT (Omp85) from Escherichia coli, the translocator domain of the autotransporter NaIP from Neisseria meningitides, FomA from Fusobacterium nucleatum, and the voltagedependent anion-selective channel, human isoform 1 (hVDACI) from mitochondria. Binding of Skp was observed for bacterial OMPs, but neither for hVDACI nor for soluble bovine serum albumin. The Skp trimer formed 1: 1 complexes, CIMP-SkP(3), with bacterial OMPs, independent of their size or origin. The dissociation constants of these OMP-SkP(3) complexes were all in the nanomolar range, indicating that they are stable. Complexes of Skp(3) with YaeT displayed the smallest dissociation constants, complexes with Na1P the largest. OMP binding to Skp(3) was pH-dependent and not observed when either Skp or OMPs were neutralized at very basic or very acidic pH. When the ionic strength was increased, the free energies of binding of Skp to OmpA or OmpG were reduced. Electrostatic interactions were therefore necessary for formation and stability of OMP-SkP3 complexes. Light-scattering and circular dichroism experiments demonstrated that Skp(3) remained a stable trimer from pH 3 to pH 11. In the OmpA.Skp(3) complex, Skp efficiently shielded tryptophan residues of the transmembrane strands of OmpA against fluorescence quenching by aqueous acrylamide. Lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, bound to OmpA.Skp(3) complexes at low stoichiometries. Acrylamide quenching of fluorescence indicated that in this ternary complex, the tryptophan residues of the transmembrane domain of OmpA were located closer to the surface than in binary OmpA.Skp(3) complexes. This may explain previous observations that folding of Skp-bound OmpA into lipid bilayers is facilitated in presence of LPS. (c) 2007 Elsevier Ltd. All rights reserved.
机译：外膜蛋白（OMPs）与来自大肠杆菌的周质伴侣Skp的相互作用尚不清楚。我们已经研究了Skp与不同起源，大小和功能的各种CIMP的结合。这些是来自大肠杆菌的OmpA，OmpG和YaeT（Omp85），来自脑膜炎奈瑟氏球菌的NaIP自转运蛋白NaIP的转运蛋白域，来自核梭菌的FomA和来自线粒体的电压依赖性阴离子选择性通道，人同工型1（hVDACI）。对于细菌OMP，未观察到Skp的结合，但对于hVDACI和可溶牛血清白蛋白均未观察到Skp的结合。 Skp三聚体与细菌OMP形成1：1复合物CIMP-SkP（3），而与它们的大小或来源无关。这些OMP-SkP（3）复合物的解离常数都在纳摩尔范围内，表明它们是稳定的。 Skp（3）与YaeT的复合​​物显示最小的解离常数，与Na1P的复合物最大。 OMP与Skp（3）的结合是pH依赖性的，当在非常碱性或非常酸性的pH下中和Skp或OMP时都没有观察到。当离子强度增加时，Skp与OmpA或OmpG结合的自由能降低。因此，静电相互作用对于OMP-SkP3复合物的形成和稳定性是必需的。光散射和圆二色性实验表明，Skp（3）在pH 3至pH 11时仍保持稳定的三聚体。在OmpA.Skp（3）络合物中，Skp有效地屏蔽了OmpA跨膜链的色氨酸残基，以防止水溶液中的荧光猝灭。丙烯酰胺。脂多糖（LPS），革兰氏阴性细菌外膜的主要组成部分，以低化学计量比与OmpA.Skp（3）结合。丙烯酰胺的荧光猝灭表明，在该三元复合物中，OmpA跨膜结构域的色氨酸残基比二元OmpA.Skp（3）络合物更靠近表面。这可以解释以前的观察结果，即在LPS的存在下，Skp结合的OmpA折叠成脂质双层很容易。 （c）2007 Elsevier Ltd.保留所有权利。

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