电化学和荧光显微镜在质子泵膜酶研究中的单脂质体测定
Summary
在这里, 我们提出了一个方案, 研究质子在单个脂质体脂质膜上的分子转移机制, 以细胞色素b3 为例。结合电化学和荧光显微镜, 可以单独检测和分析含有单一或多种酶的单囊腔内的 ph 值变化。
Abstract
电子转移链的质子泵送酶将氧化还原反应与质子在膜上的移位结合在一起, 形成用于 atp 生产的质子动力。膜蛋白的两亲性需要特别注意它们的处理, 在研究质子转移等膜转运过程时, 重组到天然脂质环境是不可或缺的。在这里, 我们详细介绍了一种方法, 已被用于研究细胞色素博3的膜氧化还原酶的质子泵送机制 , 并以大肠杆菌的细胞色素 b3 为例。电化学和荧光显微镜的结合用于控制奎宁池的氧化还原状态, 并监测腔内的 ph 值变化。由于荧光显微镜的空间分辨率, 可以同时测量数百种脂质体, 而酶含量可以缩小为单个酶或每个脂质体的转运体。相应的单酶分析可以揭示酶功能动力学中的模式, 否则可能会被整个群体的行为所隐藏。我们包括用于自动图像分析的脚本的描述。
Introduction
有关酶机制和动力学的信息通常是在集合或宏观水平上获得的, 其中酶种群在数千到数百万个分子中, 其中的测量值代表一个统计平均值。然而, 众所周知, 复杂的大分子, 如酶, 可能表现出异质性, 在他们的行为和分子机制观察到的合奏水平不一定是有效的每一个分子。这种在单个分子尺度上的偏差已经通过对单一酶的研究得到了广泛的证实, 在过去 20年中出现了各种方法1。值得注意的是, 荧光检测的单个酶活性已被用来调查酶活性2,3的异质性或发现所谓的记忆效应 (高酶活性的时期, 成功的低时期活动, 反之亦然)4,5。
许多单一的酶研究要求酶固定在表面或空间固定在另一种方式, 以保持足够长的时间在视野中进行连续观测。酶封装到脂质体已被证明能够使酶固定化, 同时防止任何负面影响, 由于表面酶或蛋白质相互作用6,7。此外, 脂质体提供了一个独特的可能性, 研究单膜蛋白在其天然脂质双层环境8,9,10.
一类膜蛋白, 转运体, 在细胞膜上运动物质的定向移位, 这种行为只有在蛋白质重组成脂质双层 (如脂质体)时才能被研究 11, 12,13。例如, 原核和真核电子转运链的几种酶所表现出的质子移位, 通过产生用于 atp 合成的质子动力, 在细胞呼吸中起着重要作用。在这种情况下, 质子泵送活动与电子转移耦合, 尽管这一过程的详细机制往往仍然难以捉摸。
最近, 我们证明了将荧光检测与电化学结合起来研究大肠杆菌末端泛醇氧化酶的质子泵作用的可能性 (细胞色素 b3)重组在脂质体14。这是通过将一种 ph 敏感的膜不透性荧光染料封装到由 e 制备的脂质体腔中来实现的.大肠杆菌极性脂质 (图 1a)。对蛋白质量进行了优化, 使大多数脂质体要么没有或只含有一个重组酶分子 (根据泊松分布)。在脂质体和溶液中的 (环境) 氧中加入泛喹酮, 提供了细胞色素 b3 的两个底物。然后, 脂质体被稀疏地吸附在半透明的超光滑金电极上, 上面覆盖着一个自组装的 6-硫代己醇单层。最后, 电极安装在一个简单的光谱电化学电池的底部 (图 1b)。对奎宁池氧化还原状态的电化学控制允许人们在任何时候灵活触发或停止酶反应, 而 ph 敏感染料则用于监测脂质体腔内的 ph 值变化, 其原因是质子通过酶。利用第二种脂质结合荧光染料的荧光强度, 可以确定单个脂质体的大小和体积, 从而定量酶质子的抽运活性。使用这种技术, 我们发现细胞色素 b3 分子能够 进入自发泄漏状态, 迅速耗散质子的动力。本文的目的是详细介绍单脂质体测量技术。
Protocol
1. 利皮/uq-10/fdll 混合物的制备 注: 用于脂质体制备的大肠杆菌脂质应在重建。 使用玻璃注射器, 将来自大肠杆菌 (25 mg/ml) 的脂质极性提取物的氯仿库存200μl 转移到玻璃瓶中, 制成5毫克的脂肪。 在脂类中加入50μml 泛素-10 (uq-10; 氯仿), 使 uq-10 的最终比例为 1:100 (1% ww)。 在脂肪/uq-10 混合物中加入波长荧光染色脂质 (fdll) 的 20μl (0.4% w w)。…
Representative Results
在每次电化学阻抗谱实验之前, 对 sam 为6mh 的镀金盖板滑移 (电极) 的质量进行了检验。图 2a显示了在吸附脂质体前后使用电化学阻抗光谱测量的具有代表性的 cole-col 图。如果 sam 的质量是足够的, 阻抗光谱应该证明一个几乎纯粹的电容行为, 导致一个半圆的科尔-科尔图。cole-cole 图形中的半圆直径等于电极表面的双层电容, 该电容应在 2.5-3.0μfcm-2范…
Discussion
该方法适用于研究呼吸膜蛋白的质子泵送, 这些蛋白质可以重组为脂质体, 并能与奎宁池交换电子。质子泵送活性可以在单酶水平上监测使用的 ph 敏感 (比例) 染料封装在脂质体腔 (图 1a)。
该方法依赖于泛喹酮 (或其他奎宁) 的能力, 纳入脂质双层, 交换电子与电极修改与 sam18。用 sam 修饰的金电极的性能非常具体。脂质体需要吸附在 sam ?…
Disclosures
The authors have nothing to disclose.
Acknowledgements
提交人感谢 bbsrc (bbp 00545″) 提供的财政支助。nh 由 vilum 基金会青年调查员方案资助。
Materials
| 6-Mercapto-1-hexanol (6MH) | Sigma | 451088 | 97% |
| 8-hydroxypyrene-1,3,6-trisulphonic acid (HPTS) | BioChemika | 56360 | |
| Aluminium holder (Electrochemical cell) | Custom-made | 30x30x7 mm; inner diameter: 26 mm; hole diameter: 15 mm | |
| Auxiliary electrode | platinum wire | ||
| Chloroform | VWR Chemicals | 83627 | |
| E.coli polar lipids | Avanti | 100600C | 25 mg/mL in chloroform |
| Epoxy | EPO-TEK | 301-2FL | low fluorescence epoxy |
| Fiji (ImageJ 1.52d) | Required plugins: StackReg and TurboReg (http://bigwww.epfl.ch/thevenaz/stackreg/) | ||
| Filter cube ("ATTO633") | Chroma Technology Corporation | Ex: 620/60 nm; DM: 660 nm; Em: 700/75 nm | |
| Filter cube ("HPTS1") | Chroma Technology Corporation | Ex: 470/20 nm; DM: 500 nm; Em: 535/48 nm | |
| Filter cube ("HPTS2") | Chroma Technology Corporation | Ex: 410/300 nm; DM: 500 nm; Em: 535/48 nm | |
| Filter cube ("Texas Red") | Chroma Technology Corporation | Ex: 560/55 nm; DM: 595 nm; Em: 645/75 nm | |
| Fluorescent dye-labelled lipids (FDLL) | ThermoFisher Scientific | T1395MP | TexasRed-DHPC was used in this work (λexc 595 nm; λem 615 nm) |
| Fluorescent dye-labelled lipids (FDLL) (alternative) | ATTO-TEC | AD 633-161 | ATTO633-DOPE can be used as alternative (λexc 630 nm; λem 651 nm) |
| Gel filtration column | GE Healthcare | 28-9893-33 | HiLoad 16/600 Superdex 75 pg, for additional protein purification |
| Glass coverslips | VWR International | 631-0172 | No 1.5 |
| Glass syringe | Hamilton | 1725 RNR | 250 µL |
| Glass vials | Scientific Glass Laboratories Ltd | T101/V1 | 1.75 mL capacity |
| Gold | GoodFellow | 99.99% | |
| Gramacidin | Sigma | G5002 | |
| Mercury sulfate reference electrode | Radiometer (Hash) | E21M012 | |
| Microcentrifuge | Eppendorf | Minispin NL040 | |
| Microscope | Nikon | Eclipse Ti | |
| Microscope Camera | Andor | Zyla 5.5 sCMOS | |
| Microscope Lamp | Nikon | Intensilight C-HGFI | |
| NIS-Elements AR 5.0.2 | Nikon | Microscope acquisition software | |
| n-Octyl β-D-glucopyranoside | Melford Laboratories | B2007 | |
| Nova 1.10 | Metrohm | Potentiostat control software | |
| Objective | Nikon | Plan Apo λ 60x/1.4 oil | |
| OriginPro 2017 | OriginLab | Plotting software | |
| O-ring (Electrochemical cell) | Orinoko | Inner diameter: 16 mm; cross section: 1.5 mm | |
| Plastic tubes | Eppendorf | 3810X | 1.5 mL |
| Polystyrene microbeads | Bio-RAD | 152-3920 | Biobeads, 20-50 mesh |
| Potentiostat | Metrohm Autolab | PGSTAT 128N | |
| Potentiostat | CH Instruments | CHI604C | |
| Scripting software | Matlab | R2017a | Required toolboxes: 'Image Processing Toolbox', 'Parallel Computing Toolbox', 'Curve Fitting Toolbox', 'System Identification Toolbox', 'Optimization Toolbox' |
| Silicon wafers | IDB Technologies LTD | Si-C2 (N<100>P) | Ø 25 mm, 525 um thick |
| Teflon cell (Electrochemical cell) | Custom-made | Outer diameter: 26 mm; inner diameter: 13.5 mm | |
| Temple-Stripped Ultra-Flat Gold Surfaces | Platypus Technologies | AU.1000.SWTSG | Alternative ready-to-use ultra-flat gold surfaces (Thickness below 100 nm on demand) |
| Thin micropipette tips | Sarstedt | 70.1190.100 | or similar gelloader tips 200 µL |
| Ubiquinone-10 | Sigma | C-9538 | |
| Ultracentrifuge | Beckman-Coulter | L-80XP | with Ti 45 rotor |
| Ultrasonic bath | Fisher Scientific | FB15063 |
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Cite This Article
Mazurenko, I., Hatzakis, N. S., Jeuken, L. J. Single Liposome Measurements for the Study of Proton-Pumping Membrane Enzymes Using Electrochemistry and Fluorescent Microscopy. J. Vis. Exp. (144), e58896, doi:10.3791/58896 (2019).