JoVE Journal > Bioengineering
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
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생체공학

构建含有G蛋白偶联受体（GPCR）的脂质膜模型

Published: February 05, 2022
doi:
10.3791/62830
, ,
1Mork Family Department of Chemical Engineering and Materials Science,University of Southern California

Summary

该协议利用琼脂糖肿胀作为一种强大且可推广的技术，用于将整体膜蛋白（IMP）掺入巨型单层脂质囊泡（GUV）中，如本文所述，用于重建人1A 5-羟色胺受体蛋白（5-HT1AR），这是药理学上重要的G蛋白偶联受体之一。

Abstract

由于质膜的复杂性以及影响活细胞中蛋白质行为的众多因素，对整体膜蛋白的结构和功能进行强有力的 体外 研究一直是一项挑战。巨型单层囊泡（GUV）是一种仿生和高度可调 的体外 模型系统，用于研究蛋白质 – 膜相互作用并以精确的刺激依赖性方式探测蛋白质行为。在该协议中，我们提出了一种廉价而有效的方法来制造GUV，其中人5-羟色胺1A受体（5-HT1AR）稳定地整合到膜中。我们使用改良的水凝胶溶胀方法制造GUV;通过将脂质膜沉积在琼脂糖和5-HT1AR的混合物上，然后对整个系统进行水润，可以将正确定向和功能性的5-HT1AR结合到膜中形成囊泡。然后，这些GUV可用于通过显微镜检查蛋白质 – 膜相互作用和定位行为。最终，该协议可以促进我们对整体膜蛋白功能的理解，提供深刻的生理洞察力。

Introduction

合成模型膜是研究生物膜基本性质和功能的强大工具。巨型单层囊泡（GUV）是研究各种质膜特性的最突出平台之一，可以设计成模仿不同的生理条件1，2，3，4，5，6，7，8。众所周知，质膜及其组织在众多细胞过程中起着关键作用，例如信号转导，粘附，内吞作用和转运9，10，11，12，13，14，15。

GUV已使用各种方法制造，包括温和的水合作用16，水凝胶溶胀17，电铸18，微流体技术19，20，21，22，喷射23和溶剂交换24，25，26。由于处理整体膜蛋白（IMP）的挑战，研究它们的 体外 平台受到限制。GUV提供了一个简化的平台，用于在模仿其原生环境的环境中研究IMP。尽管在GUV中有几种蛋白质重构的方法，但结合具有正确取向的蛋白质并保持蛋白质功能会带来挑战27。

GUV中最成功的蛋白质重构需要洗涤剂交换方法;这涉及通过洗涤剂将蛋白质从其天然环境中溶解，然后进行蛋白质纯化，然后通过各种方法用脂质代替洗涤剂分子28。虽然洗涤剂在纯化过程中用于稳定IMP的三级结构，但洗涤剂胶束对于这些蛋白质来说是一个相对不自然的环境，它们在脂质双分子层中稳定性更好，特别是对于功能研究28，29，30。此外，由于这些蛋白质的大小，细腻以及需要额外的洗涤剂交换步骤，使用传统的GUV制造技术将功能性跨膜蛋白掺入脂质双层中一直很困难27，31，32，33。使用有机溶剂去除去垢剂会导致蛋白质聚集和变性34。改进的洗涤剂介导方法一直很有希望，但是，洗涤剂去除步骤需要谨慎，并且可能需要对特定蛋白质进行优化31，35。此外，利用电铸的方法可能会限制蛋白质的选择，并且可能不适用于所有脂质组合物，特别是带电脂质31，36，37。已经使用的另一种技术是肽诱导的含有所需蛋白质的大单层囊泡（LUV）与GUV的融合，尽管它被发现是费力的，并且可能导致插入外来分子 – 梭原肽33，38，39。来自活细胞的巨型质膜囊泡（GPMV）可用于克服其中一些问题，但是它们可以对所得的脂质和蛋白质组成进行最小控制14，40，41。因此，使用我们改良的琼脂糖溶胀方法将IMPs整合到GUV的双脂层中，为在膜环境中进一步检查这些蛋白质提供了一种可靠的方法42，43，44，45。

细胞信号传导和通信涉及称为G蛋白偶联受体（GPCRs）的蛋白质家族;GPCR是最大的蛋白质家族之一，与调节情绪、食欲、血压、心血管功能、呼吸和睡眠以及许多其他生理功能有关46。在这项研究中，我们使用人类血清素1A受体（5-HT1AR），它是GPCR家族的典型成员。5-HT1AR可以在中枢神经系统（CNS）和血管中找到;它影响许多功能，如心血管，胃肠道，内分泌功能，以及参与情绪调节47。GPCR研究的一大障碍来自其复杂的两亲性结构，而GUV为研究各种感兴趣的性质提供了一个有前途的平台，包括蛋白质功能，脂质 – 蛋白质相互作用和蛋白质 – 蛋白质相互作用。已经使用各种方法来研究脂质 – 蛋白质相互作用，例如表面等离子体共振（SPR）48，49，核磁共振波谱（NMR）50，51，蛋白质脂质覆盖（PLO）测定51，52，53，54，天然质谱法55，等温滴定量热法（ITC）56，57和脂质体沉降测定法58，59。我们的实验室使用简化的GUV方法，通过包封BODIPY-GTPγS来研究脂质 – 蛋白质相互作用对蛋白质功能的影响，BODIPY-GTPγS在受体的活性状态下与Giα 亚基结合。它们的结合使荧光团解压，产生荧光信号，该荧光信号可以随着时间的推移被检测到45。此外，各种研究调查了脂质 – 蛋白质相互作用以及蛋白质在传感或稳定膜曲率中的作用60，61，利用可行的GUV方法可能是一个关键优势。

该协议展示了一种使用改性琼脂糖水凝胶系统将GPCR掺入GUV膜中的简单方法17，42。此外，基于我们之前的工作，我们的方法可能适用于可以承受短期暴露于30-40°C的IMP。 简而言之，我们铺展了一层琼脂糖薄膜与含有目标GPCR的膜片段相结合。在该层凝胶化之后，我们在琼脂糖上沉积脂质溶液，并允许溶剂蒸发。然后用水缓冲液进行系统的再水化，导致形成GUV，其中蛋白质掺入脂质双分子层。

Protocol

1. 蛋白质标记 允许NHS-罗丹明，5-HT1A 膜碎片和一个7 K MWCO自旋脱盐柱在室温下平衡。 将1mg NHS-罗丹明溶解在100μL二甲基亚砜（DMSO）中。 加入5μL1M碳酸氢钠溶液，将5-HT1AR溶液的pH值提高到pH 8。 将3.66μLNHS-罗丹明溶液加入50μL5-HT1AR溶液中，并在微量离心管中轻轻上下移液。注意：确保NHS-罗丹明的摩尔含量至少为10倍。 保持混合物避光?…

Representative Results

测量蛋白质的浓度，并将标记程度计算为染料与蛋白质之间的摩尔比为1：1。通过使用共聚焦显微镜检查GUV，我们能够确认囊泡的成功形成和蛋白质整合。用0.4摩尔%ATTO 488-DPPE标记脂质，并通过伯胺的罗丹明NHS酯修饰共价标记蛋白质。 图2a 和 图2b 分别显示了ATTO 488和罗丹明通道中掺入蛋白质的囊泡。所有显微照片都经过暗电流和平坦场校正。 <strong class…

Discussion

我们已经确定了对整体方案的成功至关重要的两个步骤：血浆处理和脂质沉积。盖玻片的血浆清洗对于确保琼脂糖水凝胶有足够的覆盖和粘附到玻璃盖玻片上至关重要。等离子清洗可以完成两件事：首先，它可以去除玻璃表面的有机物痕迹;其次，它激活盖玻片表面，随着玻璃表面亲水性的增加，润湿性增加62，63。等离子体清洁后触摸盖玻片表面会失活…

Disclosures

The authors have nothing to disclose.

Acknowledgements

我们感谢Matthew Blosser的宝贵讨论和建议。这项工作得到了海军研究办公室（N00014-16-1-2382）和国家科学基金会（PHY-1915017）的支持。

Materials

1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) Avanti Polar Lipids, Alabaster, AL 850375C-25mg
 TI-Eclipse inverted microscope Nikon, Melville, NY Eclipse Ti
1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) Avanti Polar Lipids, Alabaster, AL 850355C-25mg
13/16″ ID, 1″ OD silicon O-rings Sterling Seal & Supply, Neptune, IN 5-003-8770
16-bit Cascade II 512 electron-multiplied charge coupled device camera Photometrics, Huntington Beach, CA  Cascade II 512
1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) Avanti Polar Lipids, Alabaster, AL 850457C-25mg
50 mW solid-state lasers at 488 nm and emission filter centered at 525 nm, and 561 nm with emission filter centered at 595 nm Coherent, Santa Clara, CA 488/561-50-LS
5-HT1AR membrane fragments Perkin Elmer, Waltham, MA RBHS1AM400UA
ATTO-488-1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE) ATTO-TEC, Siegen, Germany AD 488-155
Bench top plasma cleaner Harrick Plasma, Ithaca, NY PDC-32G
bovine serum albumin (BSA) Sigma Aldrich, St. Louis, MO A9418
chloroform (CHCl3) Millipore Sigma, Burlington, MA CX1055
Cholesterol (Chol) Sigma Aldrich, St. Louis, MO C8667-5G
Corning 96-well Flat Clear Bottom Corning, Corning, NY 3904
Elmasonic E-Series E15H Ultrasonic Elma, Singen, Germany [no longer sold on main website]
glucose Sigma Aldrich, St. Louis, MO G7528
methanol (MeOH) Millipore Sigma, Burlington, MA MX0485
NanoDrop ND-1000 Thermo Fisher Scientific, Waltham, MA ND-1000
NHS-Rhodamine Thermo Fisher Scientific, Waltham, MA 46406
phosphate buffered saline (PBS) (10x PBS) Corning, Corning, NY 21-040
spinning-disc CSUX confocal head Yokogawa,Tokyo, Japan CSU-X1
standard 25 mm no. 1 glass coverslips ChemGlass, Vineland, NJ CLS-1760
sucrose Sigma Aldrich, St. Louis, MO S7903
Sykes-Moore chambers Bellco, Vineland, NJ 1943-11111
Ultra-low melting temperature agarose Sigma Aldrich, St. Louis, MO A5030
VWR Analog Heatblock VWR International, Radnor, PA [no longer sold on main website]
VWR Tube Rotator VWR International, Radnor, PA 10136-084
Zeba Spin Desalting Columns, 7K MWCO, 0.5 mL Thermo Fisher Scientific, Waltham, MA 89882
DOWNLOAD MATERIALS LIST

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GUVsGiant Unilamellar VesiclesMembrane ProteinsGPCRsG Protein-coupled Receptors5-HT1ARSerotonin ReceptorHydrogel ScaffoldLipid MembraneProtein IncorporationBiomimetic ModelIn Vitro Studies
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Elbaradei, A., Dalle Ore, L. C., Malmstadt, N. Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors (GPCRs). J. Vis. Exp. (180), e62830, doi:10.3791/62830 (2022).
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