JoVE Journal > Biochemistry
Summary
Abstract
Introduction
Protocol
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Disclosures
Acknowledgements
Materials
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Biochemistry

原子力显微镜对单蛋白质分子的力谱

Published: February 28, 2019
doi:
10.3791/55989
, , , ,
1Mechanical Engineering and Materials Science,Duke University

Summary

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。

Abstract

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。

Introduction

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。

Protocol

1. 蛋白质制备 dna 克隆。 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。 用一对限制位点分别消化质粒 pemi9…

Representative Results

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?…

Discussion

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…

Disclosures

The authors have nothing to disclose.

Acknowledgements

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。

Materials

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

  1. Rief, M., Gautel, M., Oesterhelt, F., Fernandez, J. M., Gaub, H. E. Reversible Unfolding of Individual Titin Immunoglobulin Domains by AFM. Science. 276 (5315), 1109-1112 (1997).
  2. Fisher, T. E., Oberhauser, A. F., Carrion-Vazquez, M., Marszalek, P. E., Fernandez, J. M. The study of protein mechanics with the atomic force microscope. Trends in Biochemical Sciences. 24 (10), 379-384 (1999).
  3. Ng, S., Rounsevell, R., Steward, A., Randles, L., Clarke, J. Single molecule studies of protein folding by atomic force microscopy(AFM). Abstracts of Papers of the American Chemical Society. 227, U545-U545 (2004).
  4. Rico, F., Chu, C., Moy, V. T., Braga, P. C., Ricci, D. . Methods in Molecular Biology. 736, 331-353 (2011).
  5. Muller, D. J., Dufrene, Y. F. Atomic force microscopy as a multifunctional molecular toolbox in nanobiotechnology. Nature Nanotechnology. 3 (5), 261-269 (2008).
  6. Lv, S., et al. Designed biomaterials to mimic the mechanical properties of muscles. Nature. 465 (7294), 69-73 (2010).
  7. Kim, M., et al. Nanomechanics of Streptavidin Hubs for Molecular Materials. Advanced Materials. 23 (47), 5684-5688 (2011).
  8. Gonzalez, M. A., et al. Self-Adhesive Hydrogels from Intrinsically Unstructured Proteins. Advanced Materials. , (2017).
  9. Oberhauser, A. F., Hansma, P. K., Carrion-Vazquez, M., Fernandez, J. M. Stepwise unfolding of titin under force-clamp atomic force microscopy. Proceedings of the National Academy of Sciences. 98 (2), 468-472 (2001).
  10. Li, Q., Scholl, Z. N., Marszalek, P. E. Capturing the Mechanical Unfolding Pathway of a Large Protein with Coiled-Coil Probes. Angewandte Chemie International Edition. 53 (49), 13429-13433 (2014).
  11. Davis, L. . Basic methods in molecular biology. , (2012).
  12. Scholl, Z. N., Josephs, E. A., Marszalek, P. E. A Modular, Non-Degenerate Polyprotein Scaffold for Atomic Force Spectroscopy. Biomacromolecules. , (2016).
  13. Scholl, Z. N. . The (Un) Folding of Multidomain Proteins Through the Lens of Single-molecule Force-spectroscopy and Computer Simulation. , (2016).
  14. Pawlak, K., Strzelecki, J. Nanopuller-open data acquisition platform for AFM force spectroscopy experiments. Ultramicroscopy. 164, 17-23 (2016).
  15. . Nanopuller Available from: https://sourceforge.net/projects/nanopuller/ (2018)
  16. Scholl, Z. N., Marszalek, P. E. Improving single molecule force spectroscopy through automated real-time data collection and quantification of experimental conditions. Ultramicroscopy. 136, 7-14 (2014).
  17. Bouchiat, C., et al. Estimating the persistence length of a worm-like chain molecule from force-extension measurements. Biophysical journal. 76 (1), 409-413 (1999).
  18. Su, T., Purohit, P. K. Mechanics of forced unfolding of proteins. Acta. 5 (6), 1855-1863 (2009).
  19. Steward, A., Toca-Herrera, J. L., Clarke, J. Versatile cloning system for construction of multimeric proteins for use in atomic force microscopy. Protein science. 11 (9), 2179-2183 (2002).
  20. Scholl, Z. N., Josephs, E. A., Marszalek, P. E. Modular, Nondegenerate Polyprotein Scaffolds for Atomic Force Spectroscopy. Biomacromolecules. 17 (7), 2502-2505 (2016).
  21. Hoffmann, T., et al. Rapid and Robust Polyprotein Production Facilitates Single-Molecule Mechanical Characterization of β-Barrel Assembly Machinery Polypeptide Transport Associated Domains. ACS. 9 (9), 8811-8821 (2015).
  22. Dudko, O. K., Hummer, G., Szabo, A. Theory, analysis, and interpretation of single-molecule force spectroscopy experiments. Proceedings of the National Academy of Sciences of the United States of America. 105 (41), 15755-15760 (2008).
  23. Popa, I., Berkovich, R., Alegre-Cebollada, J., Rivas-Pardo, J. A., Fernandez, J. M. Halotag Tethers to Study Titin Folding at the Single Molecule Level. Biophysical journal. 106 (2), 391a (2014).
  24. Yu, H., Siewny, M. G., Edwards, D. T., Sanders, A. W., Perkins, T. T. Hidden dynamics in the unfolding of individual bacteriorhodopsin proteins. Science. 355 (6328), 945-950 (2017).
  25. Rico, F., Gonzalez, L., Casuso, I., Puig-Vidal, M., Scheuring, S. High-speed force spectroscopy unfolds titin at the velocity of molecular dynamics simulations. Science. 342 (6159), 741-743 (2013).
  26. He, Y., Lu, M., Cao, J., Lu, H. P. Manipulating protein conformations by single-molecule AFM-FRET nanoscopy. ACS nano. 6 (2), 1221-1229 (2012).
  27. Fotiadis, D., Scheuring, S., Müller, S. A., Engel, A., Müller, D. J. Imaging and manipulation of biological structures with the AFM. Micron. 33 (4), 385-397 (2002).
  28. Edwards, D. T., Faulk, J. K., LeBlanc, M. A., Perkins, T. T. Force Spectroscopy with 9-μs Resolution and Sub-pN Stability by Tailoring AFM Cantilever Geometry. Biophysical journal. 113 (12), 2595-2600 (2017).
  29. Dudko, O. K., Mathe, J., Szabo, A., Meller, A., Hummer, G. Extracting kinetics from single-molecule force spectroscopy: Nanopore unzipping of DNA hairpins. Biophysical. 92 (12), 4188-4195 (2007).
  30. Scholl, Z. N., Li, Q., Yang, W., Marszalek, P. E. Single-molecule Force Spectroscopy Reveals the Calcium Dependence of the Alternative Conformations in the Native State of a βγ-Crystallin Protein. Journal of Biological Chemistry. 291 (35), 18263-18275 (2016).

Tags

Keywords: Single-molecule Force SpectroscopyAtomic Force MicroscopeProtein StabilityUnfolding RateUnfolding PathwayPolyproteinProtein ConcentrationCantileverAFM HeadLaserPhotodiode
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Scholl, Z. N., Li, Q., Josephs, E., Apostolidou, D., Marszalek, P. E. Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope. J. Vis. Exp. (144), e55989, doi:10.3791/55989 (2019).
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