Name: force spectroscopy of single protein molecules using an atomic force microscope
Uploaded: 2019-02-28T12:30:00
Description: Watch this Scientific Journal Video about Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope at JoVE.com

这项工作得到了国家科学基金会的支持, 将 mcc-1244297 和 mcc-1517245 提供给 pem。

1. 蛋白质制备
<ol><li>dna 克隆。<ol>
<li>合成感兴趣的 dna 序列, 例如, ni 10c10的dna 序列, 或使用标准分子生物学技术通过pcr 从宿主生物体分离11。在合成过程中或通过在 pcr 引物的 5 '-末端放置位点, 以对应于质粒 pemi91 (adgene #74888)12中的一个模块, 从而将感兴趣的基因与限制位点结合起来。</li>
		<li>用一对限制位点分别消化质粒 pemi9...

<ol>
	<li>Rief, M., Gautel, M., Oesterhelt, F., Fernandez, J. M., Gaub, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversible+Unfolding+of+Individual+Titin+Immunoglobulin+Domains+by+AFM.">Reversible Unfolding of Individual Titin Immunoglobulin Domains by AFM.</a> Science. 276 (5315), 1109-1112 (1997).</li><li>Fisher, T. E., Oberhauser, A. F., Carrion-Vazquez, M., Marszalek, P. E., Fernandez, J. 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K., Hummer, G., Szabo, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theory,+analysis,+and+interpretation+of+single-molecule+force+spectroscopy+experiments.">Theory, analysis, and interpretation of single-molecule force spectroscopy experiments.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (41), 15755-15760 (2008).</li><li>Popa, I., Berkovich, R., Alegre-Cebollada, J., Rivas-Pardo, J. A., Fernandez, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Halotag+Tethers+to+Study+Titin+Folding+at+the+Single+Molecule+Level.">Halotag Tethers to Study Titin Folding at the Single Molecule Level.</a> Biophysical journal. 106 (2), 391a (2014).</li><li>Yu, H., Siewny, M. G., Edwards, D. T., Sanders, A. W., Perkins, T. 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该协议中的一个关键步骤是使用多蛋白, 在步骤1.1.2 中描述, 它作为对 "指纹" 单分子事件的积极控制。一般来说, 必须有多蛋白蛋白的展开事件 (对于 i91, 这意味着展开的力量约 200 pn 和轮廓长度增量约28纳米), 以明确的结论, 感兴趣的蛋白质已经展开。例如, 当感兴趣的蛋白质由两边的三个 i91 域侧翼时, 则必须至少有四个 i91 事件来得出结论, 它是一个正的单分子事件。如果没有通过多蛋白或其他手...

afm 在单分子力光谱 (smfs) 方面的研究进展使单蛋白分子的机械操作和精确表征得以实现。这种表征对蛋白质力学1,2、蛋白质折叠3、蛋白质配体相互作用4、蛋白质-蛋白质相互作用5和基于蛋白质的工程产生了新的见解材料6,7,8。smfs 对于研究展开的蛋白质特别有用, 因为 afm 的拉伸允许蛋白质分子中的化学和物理键根据其刚度逐渐扩展, 从而导致轮廓长度不断增加。这种蛋白质分子的过度拉伸会在力-延伸曲线中产生突然的转变, 从而导致破裂事件 (或力峰)。力峰直接提供了在机械展开过程中蛋白质的展开力和结构变化的信息。最早使用 afm 的一项研究测量了 titin1 , 发现了蛋白质在生理条件下展开和折叠的新方面, 而不使用非自然变性物, 如浓缩化学品或极端温度。
smfs 实验是在各种仪器上进行的, 尽管在这里我们只考虑 afm。afm 由四个主要元素组成: 探头、检测器、样品支架和压电扫描仪。探头是悬臂自由晃动端的一个尖锐的尖端。校准后, 在连接分子的拉伸过程中, 使用激光束测量悬臂的弯曲, 激光束从悬臂后面反射出来, 使用 hooke 定律精确地确定力。反射激光束投射成一个象限光电二极管检测器, 该探测器产生的电压与激光束从二极管中心的位移成比例。流体中含有蛋白质样品的基板安装在三维压电级, 可通过亚纳米精度进行控制。计算机从光电二极管探测器读取电压, 并通过计算机控制的电压供应控制3d 级。这些压电致动器级通常配备电容式或应变式位置传感器, 以精确测量压电位移, 并通过反馈控制系统纠正滞后。使用对压电器进行出厂校准的压电电压常数, 将压电控制器输出的传感器信号转换为距离。图 2显示了拉实验的力-扩展曲线的一个示例。
afm-smfs 实验有两种类型: 恒速和恒力拉力测量。恒力 smfs 测量在 oberhauser等中进行了描述。9, 而在这里, 我们专注于恒定速度测量。通过向压电提供电压, 使基板相对于悬臂尖端轻轻移动, 从而完成了典型的 afm 恒速拉拔实验。一个典型的实验有尖端最初按在表面。拉动测量是通过将基板移离尖端以使其脱离接触开始的。如果一种蛋白质最初与尖端接触, 它将被拉扯, 并测量对抗位移的力量的展开痕迹。然后将底物带回与尖端接触, 并测量从力位移中确定蛋白质折叠的放松痕迹。

<table>
	<tbody>
		<tr>
			<td>AFM Specimen Discs, 15mm diameter</td>
			<td>Ted Pella, Inc.</td>
			<td>16218</td>
			<td>Serve as base for glass substrate</td>
		</tr>
		<tr>
			<td>Round Glass Coverslips, 15mm diamiter No.1 Thick</td>
			<td>Ted Pella, Inc.</td>
			<td>26024</td>
			<td>serve as glass substrate and base for gold coating</td>
		</tr>
		<tr>
			<td>Adhesive Tabs</td>
			<td>Ted Pella, Inc.</td>
			<td>16079</td>
			<td>Paste on AFM Specimen Discs to provide a sticky face for attaching glass coverslips</td>
		</tr>
		<tr>
			<td>STD Multimode head assembly</td>
			<td>Bruker Nano Inc.</td>
			<td>1B75C</td>
			<td>AFM head</td>
		</tr>
		<tr>
			<td>Glass probe holder</td>
			<td>Bruker Nano Inc.</td>
			<td>MTFML-V2</td>
			<td>Glass probe holder for scanning in fluid with the MultiMode AFM.&#160;&#160;</td>
		</tr>
		<tr>
			<td>Microlever AFM probes</td>
			<td>Bruker Nano Inc.</td>
			<td>MLCT</td>
			<td>Silicon Nitride cantilevers with Silicon Nitride tips, ideal for contact imaging modes</td>
		</tr>
		<tr>
			<td>AFM probes with Au coated tips</td>
			<td>Bruker Nano Inc.</td>
			<td>OBL-10</td>
			<td>Cantilevers for pulling on proteins with low unfolding force</td>
		</tr>
		<tr>
			<td>Multifunction Data Acquisition (DAQ) Card,16-Bit, 1 MS/s (Multichannel), 1.25 MS/s (1-Channel), 32 Analog Inputs</td>
			<td>National Instruments</td>
			<td>PCI-6259</td>
			<td>Data Acquisition for signals from AFM head and Piezo Actuators</td>
		</tr>
		<tr>
			<td>LISA Linear Piezo Stage Actuators</td>
			<td>Physik Instrumente LP</td>
			<td>P-753.11C</td>
			<td>Piezo Actuator to control the position of substrate and perform pulling measurements</td>
		</tr>
		<tr>
			<td>XY Piezo Stage</td>
			<td>Physik Instrumente LP</td>
			<td>P-541.2CD</td>
			<td>Piezo Actuator to control the position of substrate and scan on substrate surface</td>
		</tr>
	</tbody>
</table>

force spectroscopy of single protein molecules using an atomic force microscope

蛋白质从氨基酸序列到原生三维结构的折叠过程的测定是生物学中的一个重要问题。原子力显微镜 (afm) 可以通过使单个蛋白质分子的拉伸和松弛来解决这个问题, 这直接证明了特定的展开和折叠特性。基于 afm 的单分子力谱 (afm-smfs) 提供了一种方法, 可以持续测量蛋白质中的高能构象, 而这些构象在传统的体积 (生化) 测量中是不可能的。尽管发表了许多论文来展示 afm-smfs 的原理, 但由于缺乏详尽完整的协议, 进行 smfs 实验并不容易。在本研究中, 我们简要阐述了 afm 的原理, 并详细介绍了协议、程序和数据分析, 作为从 smfs 实验中取得良好效果的指导。我们演示了具有代表性的单蛋白机械展开测量的 smfs 结果, 并为一些常见的问题提供了故障排除策略。

图 2显示了此协议的代表性结果。这两个面板都显示了蛋白质的具有代表性的力延伸曲线。顶部显示的结果来自 i91 多蛋白, 而底部显示 i91 蛋白两侧的蛋白质感兴趣, ni10c分子。这些记录显示了 i91 (200 pn) 的特征力和轮廓长度增量 (28 nm), 表明 afm 的对齐和校准是成功的。然后, 这些力延伸曲线可以通过蠕虫样链 (虚线) 进行分析, 这有助于确定分?...

Watch this Scientific Journal Video about Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope at JoVE.com

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope

原子力显微镜对单蛋白质分子的力谱

我们描述了使用原子力显微镜测量单个蛋白质分子的机械性能和机械展开途径的详细程序和策略。我们还展示了代表性的结果, 作为选择和理由的良好单蛋白分子记录的参考。

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. ...

单分子力光谱学可以让我们用力操纵单个分子，从而在原子尺度上为分子生物学提供巨大的见解。在这里，我们使用原子力显微镜演示恒定速度测量。我们最终可以使用这些数据来确定蛋白质的稳定性、展开速率和展开路径。要开始该步骤，请将非导电胶粘剂卡舌连接到干净、15 毫米宽的铁盘上。取下盖板，将清洁、未涂覆或镀金的玻璃滑轨牢固地压在外露胶粘剂上。将幻灯片存放在干净、有盖的培养皿中。接下来，将纯化的多蛋白浓缩在离心过滤器中，然后反转柱，将蛋白质放入缓冲液中，用于实验。根据280纳米的吸光度确定适当的蛋白质浓度，然后准备100微升的100微升每毫升蛋白质溶液。将蛋白质溶液的 60 微升滴涂抹在幻灯片的中心。注意不要让液体在幻灯片和铁盘之间滑动，因为这将导致在实验过程中不受控制的样品运动。让样品在室温下至少坐10分钟。首先，小心地拿起悬臂，放在探针保持单元中。确保悬臂牢固地固定。然后将保持单元放在 AFM 头中。将头部置于倒置显微镜舞台上，将电池组连接到 AFM 头，为激光供电。为显微镜探测器设置摄像机，以在监视器或电视屏幕上显示激光。在显微镜的引导下，将激光定位到悬臂尖端。样品准备就绪后，将实验缓冲液的 10 微升刷新到探针保持单元的每个端口中。从样品幻灯片中抽取约 40 微升液体，并添加 40 微升缓冲液。同时，开始准备原子力显微镜进行测量。首先，将幻灯片放在压电电机上方的磁铁上。确认 AFM 级处于提升位置，然后将 AFM 头与悬臂放在样品液滴上方的舞台上。接下来，在 AFM 光电二极管前面放置一张小纸。调整激光，直到纸张上的斑点明亮且聚焦，然后取出纸张。启动 AFM 软件以查看来自光电二极管的信号。调整 AFM 头镜，使激光信号在所有四个光电二极管象限中最大化，顶部和底部象限对之间的不同信号为零。要开始校准 AFM，请将 AFM 头信号的滤波器设置设置为全带宽，并确保压电已关闭。在 AFM 软件中，测量功率谱的 512 个计算平均值，每个计算有 1，024 个数据点。将功率光谱密度集成在第一个峰值上，对应于悬臂的主要振动模式。接下来，打开压电控制器，将过滤器设置为 500 赫兹。在监控不同信号的同时，快速将 AFM 头向下移动几百微米。继续一次将头部向下移动几百微米，同时观察不同信号中的一致跳跃。当信号跳跃开始高度增加时，尖端非常接近样品表面。一旦偏转信号在尖端与表面接触时饱和，稍微抬起头部，然后调整压电电压，使 AFM 磁头向上或向下移动 100 到 5，000 纳米以找到样品表面。之后，进行一个拉拔实验，扫描大小约为500纳米，使尖端接触表面，或约100纳米的压电电机运动。使用结果计算弹簧常数和灵敏度。在软件中，将扫描大小设置为比展开的多蛋白的总理论大小大 40%。放置悬臂，使悬臂的扫描尺寸从表面的 80% 。将扫描速度设置为 300 纳米/秒。执行拉力实验并测量生成的压电位置和光电二极管信号。在整个测量过程中每小时向流体细胞添加实验缓冲液，防止样品干燥。I91多蛋白和多蛋白的录音中的展开事件，其中三个 I91 域在每侧NI10C分子的两侧，都装有蠕虫一样链模型。两个记录均显示 I91 的特征力和轮廓长度增量，表明 AFM 校准成功。含有N110C分子的多蛋白的录音显示，I91事件足以证实新事件来自感兴趣的蛋白质展开。安基林重复线平均轮廓长度约为 10.5 纳米，展开力在 8 到 25 个皮科内托恩斯之间。使用多蛋白至关重要，因为侧翼蛋白的展开事件为单个分子事件提供了指纹的正控制。如果没有这一点，数据很容易被误解。

Research

Education

JoVE Journal

JoVE Core

Cell Biology

Biochemistry

Unit 1

Visualizing Cells, Tissues, and Molecules

SCIENTISTS IN ACTION

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

固体核磁共振波谱法研究高分子组件的原子尺度结构

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the ...

科研

生物化学

Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay

用生物层干涉法捕获小分子离子通道蛋白的相互作用动力学

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

单分子力谱中蛋白质共价固定化的研究

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

细胞细胞接触物蛋白质-蛋白质相互作用的荧光波动光谱测定

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Probing The Structure And Dynamics Of Nucleosomes Using Atomic Force Microscopy Imaging

原子力显微镜成像在核细胞结构和动力学研究中的应用

Chromatin, which is a long chain of nucleosome subunits, is a dynamic system that allows for such critical processes as DNA replication and transcription ...

Imaging of Extracellular Vesicles by Atomic Force Microscopy

原子力显微镜对细胞外囊泡的成像

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane ...

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

单分子跟踪显微镜 - 确定细胞分子扩散状态的工具

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and ...

Atomic Absorbance Spectroscopy to Measure Intracellular Zinc Pools in Mammalian Cells

原子吸纳光谱测量哺乳动物细胞细胞内锌池

Transition metals are essential micronutrients for organisms but can be toxic to cells at high concentrations by competing with physiological metals in ...

Evaluation of Protein&#8211;Protein Interactions using an On-Membrane Digestion Technique

Evaluation of ProteinÃƒÂ¢Ã¢â€šÂ¬Ã¢â‚¬Å“Protein Interactions using an On-Membrane Digestion Technique

使用膜内消化技术评估蛋白质与蛋白质相互作用

Numerous intracellular proteins physically interact in accordance with their intracellular and extracellular circumstances. Indeed, cellular functions ...

Characterizing Individual Protein Aggregates by Infrared Nanospectroscopy and Atomic Force Microscopy

通过红外纳米光谱和原子力显微镜对单个蛋白质聚合进行特征化

The phenomenon of protein misfolding and aggregation results in the formation of highly heterogeneous protein aggregates, which are associated with ...