使用脚手架脂质体重构脂质近端蛋白质 - 蛋白质相互作用<em>体外</em
Summary
This paper describes a method for assessing the interactions and assemblies of integral membrane proteins in vitro with various partner factors in a lipid-proximal environment.
Abstract
体外整合膜蛋白的研究经常由疏水跨膜结构域的存在复杂。这些研究进一步复杂化，洗涤剂溶解的膜蛋白的重新纳入到脂质体是一种随机过程，其中蛋白质拓扑是无法执行。本文提供了一种替代方法，它利用基于脂质体的脚手架这些具有挑战性的技术。蛋白质溶解度是由跨膜结构域的缺失增强，并且这些氨基酸与一个系栓部分取代，如His标签。此系链与，这在脂质体的表面实施均匀的蛋白质拓扑结构（由次氮基三乙酸（NTA（镍2+））为His-标记的蛋白的协调的Ni 2+）的锚定基团相互作用。一个例子给出，其中用完整的膜蛋白，线粒体裂变因子（MFF）动力素相关蛋白1（DRP1）之间的相互作用，是英伟使用这种支架脂质体法stigated。在这项工作中，我们已经证明MFF的有效招募可溶性DRP1到脂质体的表面上，这刺激其GTP酶活性的能力。此外，DRP1能够tubulate在特定脂质的存在下MFF装饰脂质模板。本实施例说明采用结构和功能分析的脂质体的支架的有效性，并突出MFF在调节DRP1活性的作用。
Introduction
研究膜近端蛋白质-蛋白质相互作用是一个具有挑战性的努力由于在扼要涉及1的整合膜蛋白的天然环境中的困难。这是由于洗涤剂增溶的必要性和蛋白在脂蛋白体不一致取向。为了避免这些问题，我们采用的策略，从而整合膜蛋白的可溶性结构域被表达为His-标签的融合蛋白质，以及这些的可溶性片段在脂质经由相互作用锚定到支架的脂质体与NTA（镍2+）首基表面。使用这些支架，脂质 – 近侧蛋白质相互作用可以在一定范围内的脂质和蛋白质的组合物进行研究。
我们已经有效地应用这种方法来调查支配线粒体裂变复杂的装配关键蛋白 – 蛋白相互作用并检查调节这种公关脂相互作用ocess 2。期间线粒体裂变，一个保守的膜重塑的蛋白质，称为动力素相关蛋白1（DRP1）3，响应于调节能量平衡，凋亡信号等几个蜂窝信号募集至线粒体外膜（OMM）的表面整体线粒体的过程。 8 –这个大，胞浆GTP酶是通过与不可分割的OMM蛋白4次互动招募到线粒体的表面。一个这样的蛋白质的作用，线粒体裂变因子（MFF），一直难以阐明由于在体外用DRP1表观弱相互作用。然而，遗传研究清楚地表明，MFF是成功的线粒体分裂7,8至关重要。在这个手稿中描述的方法能够通过引入促进DRP1-MFF相互作用同时脂质相互作用克服了以前的缺点。总体而言，这一新的分析revea导致基本相互作用指导线粒体分裂复杂的装配和这一重要分子机器正在进行的结构和功能研究提供了一个新的阶段。
迄今为止，DRP1以及MFF之间的相互作用的研究已经通过MFF 9固有的灵活性复杂，DRP1的异质性聚合物2,10，并且难以纯化和重建全长MFF具有完整跨膜结构域11。我们通过使用NTA（镍离子 ）支架脂质体重组His标记的MFF缺乏跨膜结构域（MffΔTM-他6）解决了这些难题。这一战略是有利的，因为MffΔTM是非常易溶当E的表达。大肠杆菌 ，这种分离蛋白很容易在支架脂质体重组。当拴这些脂质模板，MFF假定在膜的表面上的相同的，面向外的取向。除了这些优点，线粒体脂质，如心磷脂，分别加入稳定MFF折叠和关联与膜11。心磷脂也与DRP1 2,12的可变结构域可稳定这种无序区和促进裂变机械的组件交互。
这种稳健的方法可广泛应用于未来的研究，设法评估近膜蛋白相互作用。通过使用附加的系链/亲和力的相互作用，这些膜重建研究的复杂性可以提高，以模仿附加在细胞内的膜的表面发现的复杂性。与此同时，脂质组合物可以被修改以更准确地模仿这些大分子复合的天然环境。总之，这种方法提供了研究蛋白质的关键的细胞PROC中的相对贡献和脂类塑造膜形态为手段S弯。
Protocol
Representative Results
Discussion
这个协议提供了调查涉及整合膜蛋白的蛋白质 – 蛋白质相互作用的方法。利用模块化脂质体脚手架，调查能够评估脂质近侧环境的一种或多种蛋白质的活性。以前的研究已经证明了质膜24的受体酶类似的方法– 26。我们扩大了这种方法纳入脂质辅因子和探索弥补线粒体分裂机械mechanoenzymatic核心蛋白之间的相互作用。
对于以上提出的模型系统中，我们发?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
The authors would like to acknowledge the funding received from the American Heart Association (SDG12SDG9130039).
Materials
| Phosphatidylcholine (DOPC) | Avanti Polar Lipids | 850375 | |
| Phosphatidylethanolamine (DOPE) | Avanti Polar Lipids | 850725 | |
| DGS-NTA(Ni2+) | Avanti Polar Lipids | 790404 | |
| Bovine Heart Cardiolipin (CL) | Avanti Polar Lipids | 840012 | |
| Chloroform | Acros Organics | 268320010 | |
| Liposome Extruder | Avanti Polar Lipids | 610023 | |
| Cu/Rh Negative Stain Grids | Ted Pella | 79712 | |
| Microfuge Tube | Beckman | 357448 | |
| GTP | Jena Biosciences | NU-1012 | |
| GMP-PCP | Sigma Aldrich | M3509 | |
| Microtiter Plate strips | Thermo Scientific | 469949 | |
| EDTA | Acros Organics | 40993-0010 | |
| Instant Blue Coomassie Dye | Expedeon | ISB1L | |
| HEPES | Fisher Scientific | BP310 | |
| BME | Sigma Aldrich | M6250 | |
| KCL | Fisher Scientific | P330 | |
| KOH | Fisher Scientific | P250 | |
| Magnesium Chloride | Acros Organics | 223211000 | |
| 4-20% SDS-PAGE Gel | Bio Rad | 456-1096 | |
| 4x Laemmli Loading Dye | Bio Rad | 161-0747 | |
| HCL | Fisher Scientific | A144S | |
| Malachite Green Carbinol | Sigma Aldrich | 229105 | |
| Ammonium Molybdate Tetrahydrate | Sigma Aldrich | A7302 | |
| Laboratory Film | Parafilm | PM-996 | |
| Uranyl Acetate | Polysciences | 21447 | |
| Tecnai T12 100 keV Microscope | FEI | ||
| Optima MAX | Beckman | ||
| TLA-55 Rotor | Beckman | ||
| Refrigerated CentriVap Concentrator | Labconico | ||
| Mastercycler Pro Thermocycler | Eppendorf | ||
| VersaMax Microplate reader | Molecular Devices |
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