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Química

生物分子有序结构由短肽的自组装形成

Published: November 21, 2013
doi:
10.3791/50946
,
1Institute of Chemistry and The Center for Nanoscience and Nanotechnology,The Hebrew University of Jerusalem

Summary

本文描述了通过自组装的自发过程中形成高度有序的基于肽的结构。该方法利用市售的肽和普通的实验室设备。这一技术可以应用到大量的各种肽，并可能导致新的基于肽的装配体的发现。

Abstract

在自然界中，复杂的功能性结构是由生物分子的自组装体在温和条件下形成的。了解控制自组装的力量和体外模拟这一过程将带来在材料科学和纳米技术等领域的重大进展。在现有的生物积木，肽具有若干优点，因为它们呈现大幅度的多样性，其在大规模的合成是直接的，并且它们可以很容易地与生物和化学实体1,2修改。几类设计的肽，如环肽，两亲性分子的肽和肽结合物自组装成有序结构在溶液中。同芳香族二肽，是一类含有需要形成有序结构，例如纳米管，球体和纤维3-8的所有分子信息短的自组装肽。种类繁多的这些肽是市售。

<p级=“jove_content”>本文提出，导致由同芳香族肽的自组装形成有序结构的过程。该协议只需要商业试剂和基本的实验室设备。此外，本文描述了一些可用于基于肽的程序集的表征的方法。这些方法包括电子和原子力显微镜和傅里叶变换红外光谱（FT-IR）。此外，演示文稿的肽的混合（coassembly）及类似结构通过该方法是“上一个珠子串”的形成。9这里介绍的协议可以潜在地适用于其它类的肽或生物构建块的，并且可以导致新的基于肽的结构的发现和更好地控制他们的集会。

Introduction

自然形成了生物分子自组装的过程有序和功能结构。了解管理这个自发过程的力量可能会导致模仿自组装体外 ，因此在材料科学10,11的区域重大进展的能力。肽，具体而言，抱很大的希望作为一个生物分子构建模块，因为他们目前大型结构的多样性，易于化学合成的，并且可以很容易地与生物和化学实体功能化。多肽自组装领域是率先由查德理和他的同事，谁表现肽纳米管的自组装由环肽交替D-和L-氨基酸12。其他成功的方法的肽组件的设计，包括线性bolaamphiphile肽5，两亲分子（AP）6，非共轭自补离子肽13，表面活性剂样肽<suP> 4,14，和二嵌段copolypeptides 15。

一个更近的方法包括短芳族肽的自组装，称为同芳香族二肽。这些肽仅包含2个氨基酸，芳香性质（ 例如苯丙氨酸，苯丙氨酸，二碳酸二叔丁酯（BOC）-苯丙氨酸-Phe的生产）7,8,16-21。由这些同芳香族肽形成的结构包括管状结构，球状，片状组件和纤维6,8,15,21-32。在某些情况下，纤维产生原纤网能够产生水凝胶33-37。这些组件已被开发用于生物传感，药物输送，分子电子学等的应用程序。38-45

本文介绍了需要的，以便开始同芳香族肽的自发自组装的实验步骤。此外，它提出了肽的过程coassembly。这个过程涉及一种以上类型的肽的自组装体单体。

我们的示范包括两种市售的肽的coassembly：将二苯基丙氨酸肽（NH 2-PHE-PHE-COOH）和它的Boc保护的类似物（的Boc-Phe-苯丙氨酸-OH）。每个肽自组装成超分子结构：的二苯基丙氨酸的肽形式的管状组件和的Boc-Phe-苯丙氨酸-OH肽自组装成任一球或纤维取决于溶剂7,17,46。我们混合两种肽在一定比率和特征将得到的组合件通过电子显微镜，力显微镜和FT-IR光谱。该方法证实，它由球形元件的直径为几微米（1-4微米），它们由细长的组件的直径为几百纳米连接（〜300-800纳米）的基于肽的结构的形成。该组件像串珠串在它们的形态，如球形结构似乎就被螺纹细长的组件。因此，我们称之为这些程序集“生物分子项链”。在“生物分子项链”可以作为一种新的生物材料作为药物输送剂或作为支架用于电子应用。此外，通向肽的自组装体的方法可用于与其它类肽和生物分子。这可能会导致更好的了解参与自组装和新的有序结构形成的势力。

Protocol

1。同芳香族二肽的自组装称量所需的肽在其冻干形式（ 例如 NH 2-PHE-PHE-OH，的Boc-Phe-苯丙氨酸-COOH）和通过在1,1,1,3,3,3 -六氟溶解肽制备的原液基-2 -丙醇（HFP）到适当的浓度（ 例如 100毫克/毫升为NH 2-PHE-PHE-OH和Boc-苯丙氨酸-苯丙氨酸-COOH）的7,17,46。 采用涡和地方在板凳上，直到肽完全溶解，溶液似乎很清楚（几分钟）混合溶液中。 稀释的?…

Representative Results

本文描述了一种形成有序结构在纳米和微米尺度由肽的自组装体的方法。为了证明这一点，我们提出简单的过程，表征了两个简单的芳香肽的coassembly（ 图1）。一个肽是NH 2-PHE-PHE-OH（二苯基丙氨酸）的肽，其能够自组装在水性溶液与纳米尺寸7中空管状结构。其他的肽是其Boc保护的类似物，的Boc-Phe-苯丙氨酸-OH。这种肽可形成纤维状结构中的乙醇17,46水溶液和球…

Discussion

总之，本文说明中，基于肽的组件可以在体外形成的容易性。该过程涉及市售的肽和溶剂，并且它自发地发生的环境条件下，当加入的极性溶剂的试管中。关键的是要使用HFP作为肽的溶剂，由于肽在其他的有机溶剂的溶解度低。此外，由于HFP的高挥发性，有必要准备新鲜原液每个实验。此外，该储备液的体积应高于10微升和溶解的多肽的转移到极性溶剂（水）应迅速进行。

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Declarações

The authors have nothing to disclose.

Acknowledgements

这项工作是由居里夫人国际重返社会格兰特和由德国和以色列基金会的支持。我们承认雅尔Razvag先生原子力显微镜分析。

Materials

Name of Material/ Equipment Company Catalog Number Comments/Description
NH2-Phe-Phe-OH Bachem G-2925.0001
Boc-Phe-Phe-OH Bachem A-3205.0005
1,1,1,3,3,3-hexafluoro-2-propanol Sigma-Aldrich 52512-100ML
Ethanol absolute (Dehydrated) AR sterile Bio-Lab Ltd. 52555 Blending with TDW for the preparation of 50% solution
Uranyl acetate Sigma-Aldrich 73943 For negative staining. It is possible to work without it.
glass cover slip Marienfeld Laboratory Glassware 110590
TEM grids Electron Microscopy Sciences FCF200-Cu-50 Formvar/Carbon 200 Mesh, Cu
Quantitive filter paper Whatman 1001055
Deuterium Oxide (D2O) Sigma-Aldrich 151882-100G 99.9 atom % D
CaF2 window PIKE Technologies 160-1212 25 mm x 2 mm window. For FT-IR measurments
AFM tips NanoScience Instruments CFMR Aspire probes, CFMR-25 series
Filter units Millipore SLGV033RS Millex-GV, 0.22 μm, PVDF, 33 mm, gamma sterilized
SEM FEI Quanta 200 ESEM
TEM FEI Tecnai T12 G2 Spirit
AFM JPK Instruments A JPK NanoWizard3
FT-IR Thermo Fisher Scientific Nicolet 6700 advanced gold spectrometer
FT-IR Purge Parker BALSTON FT-IR Purge Gas Generator model 75-52
OMNIC (Nicolet) software Thermo Nicolet Corporation For FT-IR spectra analysis
Vortex mixer Wisd Laboratory Equipment ViseMix VM
Weight Mettler Toledo NewClassic MS
Sputter coater Polaron SC7640 Sputter Coater
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Tags

Keywords: Self-assemblyBiomolecular StructuresPeptidesHomoaromatic DipeptidesNanotubesSpheresFibrilsElectron MicroscopyAtomic Force MicroscopyFT-IRCoassemblyBeads On A String
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Yuran, S., Reches, M. Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides. J. Vis. Exp. (81), e50946, doi:10.3791/50946 (2013).
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