Effects of Periplasmic Chaperones and Membrane Thickness on BamA-Catalyzed Outer-Membrane Protein Folding.
Effects of Periplasmic Chaperones and Membrane Thickness on BamA-Catalyzed Outer-Membrane Protein Folding.
复制标题
DOI：
10.1016/j.jmb.2017.09.008
复制
发表时间：
2017-11-24
影响因子：
5.6
通讯作者：
Radford SE
中科院分区：
文献类型：
--
作者：
Schiffrin B;Calabrese AN;Higgins AJ;Humes JR;Ashcroft AE;Kalli AC;Brockwell DJ;Radford SE
革兰氏阴性菌中外膜蛋白（OMP）的生物发生涉及通过周质伴侣递送至β-桶组装机器（BAM），其催化OMP插入外膜。在这里，我们研究膜厚度的影响，大肠杆菌周质伴侣Skp和SurA，和BamA，BAM复合物的中央亚基，上的折叠动力学模型OMP（tOmpA）使用荧光光谱，本地质谱，和分子动力学模拟。我们发现，尽管Skp：tOmpA复合物的nM亲和力，预折叠的BamA促进从Skp释放tOmpA。这种活性位于BamA β-桶结构域中，但当存在全长BamA时更大，表明β-桶和多肽转运相关（POTRA）结构域都是最大活性所需的。相比之下，SurA无法从Skp释放tOmpA，这为顺序伴侣模型提供了直接证据。通过改变合成脂质体中的脂质酰基链长度，我们表明BamA对较厚双层中的tOmpA折叠具有更大的催化作用，这表明BAM催化涉及降低由膜的疏水厚度施加的动力学屏障。与此相一致，分子动力学模拟表明，在膜变薄/混乱的BamA的跨膜结构域的增加是最大的在较厚的双层。最后，我们证明了BamA桶的交联不影响tOmpA在1，2-二肉豆蔻酰-sn-甘油-3-磷酸胆碱（DMPC）脂质体中的折叠动力学，这表明BamA桶和/或混合桶形成的横向门控是不需要的，至少对于体外组装的小8链OMP。BAM对OMP周质运输和折叠的机制知之甚少。BamA通过减少由膜厚度施加的动力学屏障来催化折叠。BamA蛋白脂质体促进Skp结合的tOmpA的折叠。DMPC双层中tOmpA的BamA催化折叠不需要横向门控。
The biogenesis of outer-membrane proteins (OMPs) in gram-negative bacteria involves delivery by periplasmic chaperones to the β-barrel assembly machinery (BAM), which catalyzes OMP insertion into the outer membrane. Here, we examine the effects of membrane thickness, the Escherichia coli periplasmic chaperones Skp and SurA, and BamA, the central subunit of the BAM complex, on the folding kinetics of a model OMP (tOmpA) using fluorescence spectroscopy, native mass spectrometry, and molecular dynamics simulations. We show that prefolded BamA promotes the release of tOmpA from Skp despite the nM affinity of the Skp:tOmpA complex. This activity is located in the BamA β-barrel domain, but is greater when full-length BamA is present, indicating that both the β-barrel and polypeptide transport-associated (POTRA) domains are required for maximal activity. By contrast, SurA is unable to release tOmpA from Skp, providing direct evidence against a sequential chaperone model. By varying lipid acyl chain length in synthetic liposomes we show that BamA has a greater catalytic effect on tOmpA folding in thicker bilayers, suggesting that BAM catalysis involves lowering of the kinetic barrier imposed by the hydrophobic thickness of the membrane. Consistent with this, molecular dynamics simulations reveal that increases in membrane thinning/disorder by the transmembrane domain of BamA is greatest in thicker bilayers. Finally, we demonstrate that cross-linking of the BamA barrel does not affect tOmpA folding kinetics in 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes, suggesting that lateral gating of the BamA barrel and/or hybrid barrel formation is not required, at least for the assembly of a small 8-stranded OMP in vitro. Mechanisms of OMP periplasmic transport and folding by BAM are poorly understood. BamA catalyzes folding by reducing the kinetic barrier imposed by membrane thickness. BamA proteoliposomes promote folding of Skp-bound tOmpA. Lateral gating is not required for BamA-catalyzed folding of tOmpA in DMPC bilayers.
登录
查看更多内容
影响因子：
5.5
作者：
Hess, Berk;Kutzner, Carsten;Lindahl, Erik
通讯作者：
Lindahl, Erik
影响因子：
6.4
作者：
Grabowicz M;Koren D;Silhavy TJ
通讯作者：
Silhavy TJ
DOI：
10.1126/science.aad3460
发表时间：
2016-01-08
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Bakelar J;Buchanan SK;Noinaj N
通讯作者：
Noinaj N
影响因子：
3.4
作者：
Fleming, Patrick J.;Patel, Dhilon S.;Im, Wonpil
通讯作者：
Im, Wonpil
DOI：
10.1073/pnas.1415955112
发表时间：
2015-02-17
影响因子：
11.1
作者：
Hagan, Christine L.;Wzorek, Joseph S.;Kahne, Daniel
通讯作者：
Kahne, Daniel