从构造到晶体 - 迈向β桶外膜蛋白的结构解析
Summary
β-barrel outer membrane proteins (OMPs) serve many functions within the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Here, we hope to alleviate a known bottleneck in structural studies by presenting protocols for the production of β-barrel OMPs in sufficient quantities for structure determination by X-ray crystallography or NMR spectroscopy.
Abstract
Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% percent of known structures. There are two types of membrane proteins, α-helical and β-barrel. While α-helical membrane proteins can be found in nearly all cellular membranes, β-barrel membrane proteins can only be found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. One common bottleneck in structural studies of membrane proteins in general is getting enough pure sample for analysis. In hopes of assisting those interested in solving the structure of their favorite β-barrel outer membrane protein (OMP), general protocols are presented for the production of target β-barrel OMPs at levels useful for structure determination by either X-ray crystallography and/or NMR spectroscopy. Here, we outline construct design for both native expression and for expression into inclusion bodies, purification using an affinity tag, and crystallization using detergent screening, bicelle, and lipidic cubic phase techniques. These protocols have been tested and found to work for most OMPs from Gram-negative bacteria; however, there are some targets, particularly for mitochondria and chloroplasts that may require other methods for expression and purification. As such, the methods here should be applicable for most projects that involve OMPs from Gram-negative bacteria, yet the expression levels and amount of purified sample will vary depending on the target OMP.
Introduction
β桶的OMPs只能在线粒体，叶绿体外膜中找到，和革兰氏阴性菌1-3。而他们服务相似的角色为α螺旋的蛋白质，它们有非常不同的褶皱包括中央膜包埋β桶结构域范围从8-26反平行β链与各链被紧密连接到两个相邻的股线的（ 图1和2）。的β桶结构域的第一个和最后一个链，然后与另一个在反平行的方式进行交互，几乎完全（除线粒体VDAC），以关闭和从周围膜密封β桶结构域。所有β桶的OMPs具有不同序列和长度的胞外环从而起到配体的相互作用和/或蛋白质 – 蛋白质接触中起重要作用，与这些环有时是75个残基，如在奈瑟氏菌转发现结合亲大蛋白A（TBPA）4。 β桶外膜蛋白也可以有哪些作为其他域的蛋白质的功能性用途的N端或C端周质的扩展（ 例如，巴马5-7，FimD 8,9，法德勒10）。而存在11许多类型的β桶的OMPs的，两种较常见的类型在下面描述为用于那些不太熟悉的领域中，（1）的TonB依赖性转运和（2）autotransporters例子。
的TonB依赖性转运（ 例如，FEPA，TBPA，BtuB，CIR， 等 ）是用于营养进口基本与包含一个N-末端插头域选自由被发现夹着内侧的22链β-C-末端〜150个残基的桶域嵌入到外膜12（ 图3）。虽然这种插头域防止基板从自由穿过枪管域，底物结合诱导插头域第内的构象变化在导致孔形成（通过插头重排或插头的部分/完整喷射）然后可以促进跨外膜衬底输送到周质。的TonB依赖性转运是革兰氏阴性细菌的一些致病菌株的存活特别重要，如已经进化出劫持营养素如来自人类宿主蛋白质4,13,14铁直接专门转运脑膜炎奈瑟氏球菌 。
Autotransporters属于革兰氏阴性菌的V型分泌系统，是指带有一个β桶结构域（通常为12-链与ESTA和ESPP）的是任一分泌或提出了在β-桶外膜蛋白和一位乘客域单元15,16（ 图3）的表面上。这些β桶的OMPs往往成为在细胞存活和毒力的重要作用与服务于乘客域或者作为蛋白酶，粘附，和/或其它EFfector介导的发病机制。
结构的方法，如X射线晶体学，核磁共振光谱，和电子显微镜（EM）允许我们确定在原子分辨率可反过来用于破译它们外膜内究竟如何发挥作用的外膜蛋白的模型。然后可用于药物和疫苗的开发，如果适用本宝贵信息。例如，转铁蛋白结合蛋白A（TBPA）被奈瑟氏球菌的表面上发现的和所需的发病机制，因为它直接结合人转铁蛋白，然后提取并导入铁为自身的生存。无TBPA， 奈瑟不能从人类宿主清除铁和呈现非致病。势必TBPA 4人转铁的晶体结构得到解决之后，它成为更清晰的两种蛋白质如何相关，哪些区域TBPA介导的相互作用，什么残基为铁提取重要由TBPA，和怎么一会开发针对脑膜炎奈瑟氏疗法针对跨界保护区。因此，给定的β桶的OMPs的革兰氏阴性菌的生存和发病机制中的重要性，以及在线粒体和叶绿体功能，以及需要了解这个唯一的类膜蛋白的额外的结构信息，并且其中它们的功能的系统，一般的协议都带有表达和结构方法在高位纯化的目标外膜蛋白为特征的总体目标。
Protocol
Representative Results
Discussion
β桶的OMPs服务于革兰氏阴性菌，线粒体和叶绿体重要作用，并且用于结构分析，提供了大量关于在这些相应的细胞器的外膜基本分子机制信息的重要目标。然而，对于结构分析产生足够样品并不总是直截了当，因此，一般的管道提出了生产足够数量的目标β桶的OMPs结构测定的，详细说明从构建体晶体的过程。虽然这些协议已被测试，发现从革兰氏阴性细菌最外膜蛋白工作，有其局限性，有一些目…
Divulgations
The authors have nothing to disclose.
Acknowledgements
We would like to thank Herve Celia of the CNRS for providing the UV images and Chris Dettmar and Garth Simpson in the Department of Chemistry at Purdue University for providing the SONICC images. We would like to acknowledge funding from the National Institute of Diabetes and Digestive and Kidney Diseases and the Intramural Research Program at the National Institutes of Health. Additionally, we would like to acknowledge additional funding from the National Institute of General Medical Sciences (A.M.S. and C.J.), National Institute of Allergy and Infectious Diseases (N.N. 1K22AI113078-01), and the Department of Biological Sciences at Purdue University (N.N.).
Materials
| Crystallization Robot | TTP Labtech, Art Robbins | – | Any should work here, except for LCP crystallization |
| PCR thermocycler | Eppendorf, BioRad | – | |
| Media Shaker | New Brunswick, Infors HT | – | |
| UV-vis spectrometer | Eppendorf | – | |
| SDS-PAGE apparatus | BioRad | 1645050, 1658005 | |
| SDS-PAGE and native gels | BioRad, Life Technologies | 4561084, EC6035BOX (BN1002BOX) | |
| AkTA Prime | GE Healthcare | – | |
| AkTA Purifier | GE Healthcare | – | |
| Microcentrifuge | Eppendorf | – | |
| Centrifuge (low-medium speed) | Beckman-Coulter | – | |
| Ultracentrifuge (high speed) | Beckman-Coulter | – | |
| SS34 rotor | Sorvall | – | |
| Type 45 Ti rotor | Beckman-Coulter | – | |
| Type 70 Ti rotor | Beckman-Coulter | – | |
| Dounce homogenizer | Fisher Scientific | 06 435C | |
| Emulsiflex | Avestin | – | |
| Dialysis tubing | Sigma | D9652 | |
| LCP tools | Hamilton, TTP Labtech | – | |
| VDX 24 well plates | Hampton Research | HR3-172 | |
| Sandwich plates | Hampton Research, Molecular Dimensions | HR3-151, MD11-50 (MD11-53) | |
| Grace Crystallization sheets | Grace Bio-Labs | 875238 | |
| HiPrep S300 HR column | GE Healthcare | 17-1167-01 | |
| Q-Sepharose column | GE Healthcare | 17-0510-01 | |
| Crystallization screens | Hampton Research, Qiagen, Molecular Dimensions | – | |
| Gas-tight syringe (100 mL) | Hamilton | ???? |
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