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Abstract
Introduction
Protocol
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생화학

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

Published: September 12, 2019
doi:
10.3791/60108
, , , ,
1Centre for Misfolding Diseases, Department of Chemistry,University of Cambridge, 2Institute of Biotechnology, Life Sciences Center,Vilnius University, 3Cavendish Laboratory, Department of Physics,University of Cambridge

Summary

我们描述了红外纳米光谱学和高分辨率原子力显微镜的应用，将蛋白质自组装过程可视化为寡聚体和淀粉样纤维蛋白，这与发病和发展密切相关广泛的人类神经退行性疾病。

Abstract

蛋白质误折叠和聚集的现象导致形成高度异质蛋白聚集体，这些聚合与神经退行性疾病（如阿尔茨海默氏症和帕金森病）相关。特别是低分子量聚合物，淀粉样寡聚物，已被证明具有一般细胞毒性，并被牵连为神经毒素在许多形式的痴呆症。我们说明了使用基于原子力显微镜（AFM）的方法，以解决这些骨料的形态、结构和化学特性的特性这一具有挑战性的任务，这些特性很难使用传统的结构来研究。方法或散装生物物理方法，因为它们的异质性和瞬态性。扫描探针显微镜方法现在能够研究淀粉样粒体具有亚纳米分辨率的形态。我们在这里表明，红外（IR）纳米光谱（AFM-IR），同时利用AFM的高分辨率和红外光谱的化学识别能力，可以更进一步，使个体的结构特性的表征蛋白质聚合，从而提供对聚合机制的见解。由于我们描述的方法也可以应用于研究蛋白质组件与小分子和抗体的相互作用，它可以提供基本信息，以开发新的治疗化合物来诊断或治疗神经退行性疾病。

Introduction

目前，全世界有超过4000万人受到神经退行性疾病的影响，如阿尔茨海默氏症（AD）1和帕金森氏症（PD）2型疾病。 更一般地，超过五十种病理学在分子水平上与蛋白质误折叠和聚集有关，这一过程导致不溶性纤维蛋白聚集物的增殖，称为淀粉样矿床3， 4.神经退化的分子起源及其与蛋白质形成蛋白的构象变化的联系，然而，仍然不清楚，这在很大程度上是由于异质性、瞬态性和纳米尺度的高水平病理聚合体4，5的尺寸。

在过去的几十年中，蛋白质结构的研究非常成功，广泛基于批量方法的使用，包括X射线晶体学、低温电子显微镜和核磁共振光谱学5，6,7,8,9.在这类技术中，红外（IR）光谱技术已成为一种敏感的分析工具，可以揭示蛋白质8等生物系统的化学性质。红外方法允许在蛋白质误折叠和聚合期间对蛋白质二次和四元结构进行定量。此外，为了在微观层面上进一步破译蛋白质聚合过程中复杂的自由能量景观所涉及的机械细节，一个重大进展是开发化学动力学工具，以扩展到复杂的自组装途径包括淀粉样纤维蛋白形成5，6，7，10，11，12。然而，散装光谱方法只提供关于溶液中存在的物种的异质组合或涉及特定微观步骤的平均信息，从而对个体的生物物理特性进行了调查在纳米级13、14级挑战的聚合物种。

在过去的几十年中，出现了几种显微镜技术，其操作能力小于光的衍射极限。此类方法包括电子显微镜 （EM） 和原子力显微镜 （AFM）。扫描电子显微镜 （SEM） 和传输电子显微镜 （TEM） 提供标本的二维 （2D） 图像，而 AFM 在过去几十年中已成为一种功能强大且用途广泛的技术，用于研究三维 （3D） 形态，如以及亚纳米分辨率13、14、15、16、17、18、19的样品的纳米力学特性。20,21,22,23,24,25,26,27.研究通过AFM的蛋白质聚集的理由是，这种方法能够研究溶液13、14、16中个别物种的形态。 17，19，20，21，25，27，28，29，30 31，32，33，34，35，36，37。特别是，通过监测样本作为时间的函数，AFM允许调查样本内物种形态的演变，从而能够跟踪和可视化淀粉样蛋白形成的途径23。 25，38，39，40，41，42。此外，AFM 能够量化结构参数，如溶液13、19、30、31中存在的单个物种的横截面高度和长度 ，32，33，34，35，36，37，40，43，44,45,46,47,48.然而，在研究异质和复杂的生物系统时，对单一生物物理特性的研究，例如形态学，往往是不够的。AFM、SEM 或 TEM 成像方法本身并不能轻易揭示纳米尺度上异构淀粉样聚合体的化学性质。

随着红外纳米光谱（AFM-IR）24、26、24、26、24、26 、24、26 、24、26、24、26、26 、26 、26 、26 、26 、26 、26 、26、26、24、24、26、24、26、26、26、24、26、26、24、26、26、24、26、24、26、26、24、26、24、26、26、26、24、26号蛋白质聚集领域的开发 38，42，49，50，51，52.这种创新方法利用了AFM（±1~10nm）的空间分辨率与红外化学分析能力的结合。AFM-IR 技术基于红外激光驱动的光热感应共振效应的测量，以及 AFM 尖端所调查样品的热膨胀的测量。样品可以直接由红外激光从顶部或底部在总内部反射中照亮，类似于传统的红外光谱24、42、52、53.红外激光器的典型频率可按数百千赫（1~1000 kHz）的顺序进行脉冲，并在宽光谱范围内进行调谐，通常在1000~3300厘米-1之间。虽然激光源的直径为 ±30 μm，但 AFM-IR 技术的空间分辨率名义上由 AFM 尖端直径确定，该直径可检测系统的局部热膨胀。AFM-IR 非常适合研究生物样品，因为红外信号与其厚度成正比，高达 1~1.5 μm，并且生成的红外光谱通常与相应的 FTIR 透射光谱13、54 一致 ， 55.因此，在光谱学中可以容易地应用既定的分析方法，例如通过第二导数分析研究化学变化、带形变化和去卷积。总体而言，AFM 的空间分辨率与红外光谱的化学识别能力相结合，使样品能够在纳米尺度上同时采集各种形态、机械和化学特性。

在这里，我们演示了蛋白质聚合过程的表征方案，该协议利用体外荧光测定、高分辨率 AFM 成像和纳米级 AFM-IR 的组合。这种组合方法在研究蛋白质聚合形成的单个微液滴的化学和结构特性、液-液蛋白相分离的研究以及在纳米尺度23、26、38、45、50、53， 56，57.

Protocol

1. 荧光板读取器上的聚合测定 注：此处描述的协议是如何通过化学动力学研究任何蛋白质或肽的聚合的示例。特别是，它描述了一个优化的协议，以研究A+42肽的聚合，它参与阿尔茨海默氏病的发病和进展58，59。类似的方案可以调整和采用，以研究任何蛋白质或肽的聚合。 通过电离交换和大小排除色谱技术获得A+42的超纯单?…

Representative Results

A_42聚合的代表性时间过程，由ThT荧光测定测量，如图1所示。聚合过程通常以 sigmoidal 曲线为特征，其中最初观察到滞后阶段，然后是陡峭的生长阶段，在达到平衡稳定状态达到6、7时曲线达到稳定状态之前,58.必须确保使用优化的聚合协议来生成高质量的数据，以研究与聚合过程有关的分子细节<sup class="xr…

Discussion

该协议的第一个关键步骤是制备单体蛋白，如步骤1.1和1.2中描述的A_42溶液。从高度纯、单一的溶液启动聚合过程至关重要，因为寡头或聚合物种的存在可能导致聚合动力学58的可重复性差，并诱导 AFM 中的人工制品测量（例如，纤维素物种在聚集的初始阶段将明显），这可能导致对数据的误解。基于ThT荧光测定的淀粉样形成的高度可重复动力学数据，与化学动力学5、6、7…

Disclosures

The authors have nothing to disclose.

Acknowledgements

作者感谢瑞士国家科学基金会（SNF）的财政支持（赠款号P2ELP2_162116和P300P2_171219），达尔文学院，伊拉斯谟®计划的财政支持（赠款号2018-1-LT01-KA103-046719-15400-P3）和导致这些结果的研究通过ERC赠款PhysProt（协议号337969）、纽曼基金会（T.P.J.K.）和剑桥错折疾病中心（C.G.、M.V.和T.P.J.K.）。

Materials

AFM-IR system Anasys Instruments nanoIR 2 or 3 Systems to measure thermal expansion in contact and resonance mode
Corning 96-well Half Area Black/Clear Bottom Polystyrene NBS Microplate Corning 3881
Corning Microplate Aluminium Sealing Tape Corning 6570
Double Sided Adhesive Discs AGAR Scientific AGG3347N
FLUOstar Omega BMG Labtech 415-101 Platereader
Mica Disc 10mm V1 AGAR Scientific AGF7013
Park NX10 AFM system Park Systems N/A Atomic Force Microscope
Platypus Ultra-Flat Gold Chips Platypus Technologies AU.1000.SWTSG
PPP-NCHR-10 cantilevers Park Systems PPP-NCHR-10
Protein LowBind Tubes, 2.0mL Eppendorf 30108132
Silicon gold coated cantilevers Anasys Instruments PR-EX-nIR2
SPM Specimen Discs 12mm AGAR Scientific AGF7001
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Protein AggregatesInfrared NanospectroscopyAtomic Force MicroscopySingle Molecule MethodsBiomolecular ProcessesNano ScaleHeterogeneous SystemsProtein AggregationBiomaterialsDrug DeliveryAFM ParametersAFM ImagingAFM CantileverAFM Laser BeamAFM Deflected BeamAFM OscillationAFM Resonance
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Ruggeri, F. S., Šneideris, T., Chia, S., Vendruscolo, M., Knowles, T. P. J. Characterizing Individual Protein Aggregates by Infrared Nanospectroscopy and Atomic Force Microscopy. J. Vis. Exp. (151), e60108, doi:10.3791/60108 (2019).
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