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Biochimica

聚合物拥挤脂质膜上的单分子扩散和组装

Published: July 19, 2022
doi:
10.3791/64243
, , , , ,
1Department of Chemical Engineering,Indian Institute of Science, 2Holonyak Micro & Nanotechnology Lab,University of Illinois, Urbana-Champaign, 3Center for BioSystems Science and Engineering,Indian Institute of Science

Summary

在这里，提出了一种使用单分子全内反射荧光（smTIRF）显微镜执行和分析单分子在人工拥挤脂质膜上的结合，迁移率和组装的协议。

Abstract

细胞膜是生物分子反应和信号传导的高度拥挤的环境。然而，大多数探测蛋白质与脂质相互作用的 体外 实验都采用裸双层膜。这种系统缺乏膜包埋蛋白和聚糖拥挤的复杂性，并且排除了在细胞膜表面遇到的相关体积效应。此外，形成脂质双层的带负电荷的玻璃表面可防止跨膜生物分子的自由扩散。在这里，我们提出了一种表征良好的聚合物脂质膜作为拥挤脂质膜的模拟物。该协议利用聚乙二醇（PEG）共轭脂质作为将拥挤者掺入支持的脂质双层（SLB）的通用方法。首先，介绍了用于进行单分子实验的显微镜载玻片和盖玻片的清洁程序。接下来，讨论了表征PEG-SLBs的方法，并使用单分子跟踪和光漂白进行生物分子结合，扩散和组装的单分子实验。最后，该协议演示了如何通过单分子光漂白分析监测拥挤脂质膜上细菌孔形成毒素溶细胞素A（ClyA）的纳米孔组装。还包括带有示例数据集的 MATLAB 代码，以执行一些常见分析，例如粒子跟踪、提取扩散行为和亚基计数。

Introduction

细胞膜是高度拥挤和复杂的系统1。分子拥挤会对蛋白质和脂质2，3，4等膜结合实体的扩散产生相当大的影响。同样，脂质膜上的双分子反应如受体二聚化或膜复合物的寡聚化也受到拥挤5，6，7的影响。拥挤器的性质、构型和浓度可以通过多种方式控制膜结合、扩散率和蛋白质-蛋白质相互作用8，9。由于控制细胞膜上的膜拥挤并解释其对嵌入生物分子的影响具有挑战性，因此研究人员试图建立替代体外系统10。

人工拥挤膜的一种流行方法是用聚合物（如聚乙二醇，PEG）接枝脂质11，12掺杂双层膜。在支撑脂质双层（SLB）上蛋白质和脂质动力学的可视化过程中，这些聚合物通过有效地将双层从底层载体上抬起，另外将膜包埋的组分与底层带负电的基底（如玻璃）隔离开来。通过改变聚合物的大小和浓度，可以控制分子拥挤的程度，以及它与底层固体载体的分离13，14。这显然比在没有聚合物垫15，16的固体底物上支撑的脂质双层更具优势，其中跨膜生物分子可以失去其活性17，18，19。更重要的是，它使我们能够在体外概括细胞膜的拥挤环境，这对于许多膜过程至关重要。

膜上的表面接枝聚合物也会根据其接枝密度12而改变其构型。在低浓度下，它们保持在膜表面上方的熵卷曲结构中，称为蘑菇。随着浓度的增加，它们开始相互作用并趋向于展开和延伸，最终在膜上产生密集的刷状形成21。由于从蘑菇到刷状状态的过渡是高度异质的，并且表现为聚合物的表征条件不佳，因此在聚合物接枝膜上使用表征良好的拥挤条件非常重要。与最近的一项研究20相比，我们确定并报告了维持跨膜生物分子扩散运输和活性的拥挤膜组成。

在该协议中，我们讨论了如何生成聚乙二醇化脂质膜，并为模拟两种不同聚合物配置（即蘑菇和刷子）中的拥挤的PEG密度提供了建议。该协议还描述了嵌入这些拥挤膜中的分子的单分子结合，颗粒跟踪和光漂白数据采集和分析。首先，我们描述了彻底的清洁步骤，成像室的组装以及PEG-SLB的生成。其次，我们提供了单分子结合，粒子跟踪和光漂白实验的详细信息。第三，我们讨论i）提取相对结合亲和力，ii）表征分子扩散，以及iii）从膜上的单分子电影中计数蛋白质组装中的亚基。

虽然我们用单分子成像表征了该系统，但该协议对于所有有兴趣了解拥挤对脂质膜生物分子反应的影响的膜生物物理学家都是有用的。总体而言，我们提出了一个强大的管道，用于制造拥挤和支持的脂质双层，以及对其进行的各种单分子测定和相应的分析程序。

Protocol

1. 清洁单分子实验的载玻片和盖玻片 在组装成像室之前，清洁并准备盖玻片和载玻片。使用带有金刚石涂层钻头（直径 0.5-1 毫米）的钻孔机在载玻片上钻多对孔。如果使用亚克力板，请使用激光切割机打精确的孔（0.5毫米），如图 1所示。注意：每对孔将充当单个微流体室的流量交换的入口和出口。亚克力幻灯片孔的代表性CAD文件可在 补充编?…

Representative Results

监测ClyA蛋白在聚乙二醇化膜上的结合在步骤4.5之后，通过绘制随时间与膜表面结合的颗粒数量来估计结合动力学（视频1）。当ClyA蛋白与具有5摩尔%PEG2000脂质的膜结合时，颗粒密度增加并达到饱和（图5）。与结合颗粒（青色圆圈）的指数衰减拟合给出了膜结合的时间常数（τb）（值得注意的是，在这种情况下，初始时间点[红色圆圈]不适…

Discussion

在这里，我们展示了在支持的脂质双层（SLB）上的单分子实验，这些实验表现出膜嵌入生物分子的拥挤环境。拥挤的环境产生排除体积效应，导致生物分子反应的增强1，2，39，40。对于PEG-脂质系统，其中聚合物主要占据双层外的体积，这种效应对于具有大外结构域的分子物种尤其明显。因此，与亲脂…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

作者感谢Benjamin Schuler教授分享了ClyA蛋白的表达质粒。这项工作得到了人类前沿科学计划（RGP0047-2020）的支持。

Materials

2.5 ml Syringes HMD Healthcare Dispo Van, 2.5 ml Tuberculin Plastic syringe
Acetone Finar Chemicals 10020LL025
Acrylic Sheet 2 mm thick
Acrylic Sheet BigiMall 2 mm, Clear
Bath Sonicator Branson CPX-1800
Calcium Chloride
Chloroform Sigma 528730  HPLC grade
Cholesterol Avanti 700100
Coplin Jar Duran Wheaton Kimble S6016 8 Slide Jar with Glass Cover
Coverslips VWR 631-1574 24 mm X 50 mm
Cy3-DNA Strand IDT GCTGCTATTGCGTCCGTTTGGTT
GGTGTGGTTGG-Cy3
Cyanine Dye (Cy3) Cytiva Life Sciences PA23001
DiI Invitrogen D3911 Dil Stain (1,1'-Dioctadecyl-3,3,3',3'-Tetramethylindocarbocyanine Perchlorate ('DiI'; DiIC18(3)))
DNA Connector Strand 1 Sigma Aldrich GCTGCTATTGCGTCCGTTTAGCT
GGGGGAGTATTGCGGAGGAAGC
T
DNA Connector Strand 2 Sigma Aldrich CGGACGCAATAGCAGCTCACAG
TCGGTCACAT
DNA Tocopherol Strand Biomers Toco-CCCAATGTGACCGACTGTGA
DOPE-PEG2000 Avanti 880130 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (ammonium salt)
Double Sided Tape 3M LF93010LE
Drill Bits (Diamond Coated) 0.5 – 1 mm
Drilling Machine Dremel 220 Workstation
EMCCD Andor DU-897U-CS0-#BV
Fluorescence Beads Invitrogen F10720
Glass Slides Blue Star Micro Slides, PIC-1
Glass Vials Sigma 854190
Hydrogen Peroxide Lobachemie 00182 30% Solution, AR Grade
Labolene Thermo-Fischer Scientific  Detergent
Laser 532 nm Coherent Sapphire
Laser Cutter Universal Laser Systems ILS12.75
Lissamine Rhodamine DOPE Avanti 810150 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (ammonium salt)
Methanol Finar Chemicals 30932LL025
Microscope Olympus IX81
Phosphate Buffer Saline (PBS) 1X
Plasma Cleaner Harrick Plasma Inc PDC-002
POPC Avanti 850457 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine
Programmable Syringe Pump New Era Pump Systems NE1010 High Pressure Syringe Pump
PTFE Caps Sigma 27141
PTFE Tubing Cole-Parmer WW-06417-21 Masterflex, 0.022" ID x 0.042" OD
Sulphuric Acid SD Fine Chemicals 98%, AR Grade
TIRF Objective Olympus UPLAPO100XOHR
Vacuum Desiccator Tarsons
Vortex Mixer Tarsons
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Tags

Single-molecule ImagingPolymer-crowded Lipid MembranesSupported Lipid BilayersMembrane Biomolecular ReactionsDiffusionAssemblyPOPCDOPE-PEG(2000)Small Unilamellar VesiclesSonicationMicrofluidic Imaging Chambers
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Maurya, S., Rai, V. H., Upasani, A., Umrao, S., Parwana, D., Roy, R. Single-Molecule Diffusion and Assembly on Polymer-Crowded Lipid Membranes. J. Vis. Exp. (185), e64243, doi:10.3791/64243 (2022).
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