JoVE Journal > Biochemistry
Summary
Abstract
Introduction
Protocol
Risultati
Discussion
Divulgazioni
Acknowledgements
Materials
Riferimenti
Traduzione automatica
Please note that all translations are automatically generated. Click here for the English version.
Biochimica

OaAEP1介导酶合成和聚合蛋白固定，用于单分子力光谱

Published: February 05, 2020
doi:
10.3791/60774
, , , , , ,
1State Key Laboratory of Coordination Chemistry, School of Chemistry and Chemical Engineering,Nanjing University

Summary

在这里，我们提出了一个方案，通过酶形成蛋白质聚合物与受控序列结合蛋白质单体，并将其固定在表面进行单分子力光谱研究。

Abstract

近年来，化学和生物结合技术发展迅速，使蛋白质聚合物得以形成。然而，受控的蛋白质聚合过程始终是一个挑战。在这里，我们开发了一种酶方法，用于在合理控制的序列中逐步构建聚合蛋白。在这种方法中，蛋白质单体的C端是NGL用于蛋白质结合使用OaAEP1 （奥德兰尼亚亲基苯甲酰二甲苯肽酶） 1），而N端是一个可夹紧的TEV（烟草蚀刻病毒）裂解位点加L（ENLYFQ/GL）用于临时N端保护。因此，OaAEP1一次只能添加一个蛋白质单体，然后TEV蛋白酶在Q和G之间将N-终点板分刻，以暴露NH 2-Gly-Leu。然后，该装置已准备好进行下一次 OaAEP1 结扎。工程多蛋白通过展开单个蛋白质域使用原子力显微镜为基础的单分子力光谱（AFM-SMFS）进行检查。因此，本研究为多蛋白工程和固定化提供了一个有用的策略。

Introduction

与合成聚合物相比，天然多域蛋白具有统一的结构，具有控制良好的数量和子域1的类型。此功能通常导致改善生物功能和稳定性2，3。许多方法，如基于半胱氨酸的二硫化物结合和重组DNA技术，已经开发用于建立这种聚合蛋白与多个领域4，5，6，7。然而，前一种方法总是导致一个随机和不受控制的序列，而后者会导致其他问题，包括有毒和大型蛋白质的过度表达和复合蛋白与辅因子和其他微妙酶的纯化的困难。

为了迎接这一挑战，我们开发了一种酶法，利用蛋白胶合OaAEP1与蛋白酶TEV8、9相结合，将蛋白质单体结合在一起，以循序渐进的方式用于聚合物/多蛋白。OaAEP1是一种严格而高效的内肽酶。如果N-终点为Gly-Leu残留物（GL），则两种蛋白质可于30分钟内通过OaAEP1通过两个终点线（NGL）进行共价连接，而C-终点为NGL残留物10。然而，使用OaAEP1只将蛋白质单体与蛋白质聚合物联系起来，其序列与基于半胱氨酸的耦合方法一样不受控制。因此，我们设计了具有可移动TEV蛋白酶位点的蛋白质单元的N-终点，以及作为ENLYFQ/G-L-POI的亮氨酸残留物。在 TEV 裂解之前，N 终端不会参与 OaAEP1 连接。然后，N-总站的GL残留物，与进一步的OaAEP1结扎兼容，在TEV裂解后暴露。因此，我们实现了多蛋白的连续酶生物合成方法，序列控制相对较好。

在这里，我们的分步酶合成方法可用于多蛋白样品制备，包括序列控制和不受控制，以及蛋白质固定用于单分子研究，特别是对于复杂系统，如金属蛋白。

此外，基于AFM的SMFS实验使我们能够确认蛋白质聚合物在单分子水平上的结构和稳定性。单分子力光谱，包括AFM，光学钳子和磁钳，是纳米技术中一般工具，以机械方式操纵生物分子，测量其稳定性11，12，13，14，15，16，17，18，19，20。单分子AFM已广泛应用于蛋白质（非）折叠21，22，23，24，25，受体-配体相互作用的强度测量26，27，28，29，30，31，32，33，34， 35，无机化学键20，36，37，38，39，40，41，42，43和金属配体键在金属蛋白44，45，46，47，48，49，50.在这里，使用单分子AFM验证基于相应蛋白质展开信号的合成多蛋白序列。

Protocol

1. 蛋白质生产 基因克隆 购买感兴趣的蛋白质（POI）的基因编码：泛素、鲁布雷多辛（RD）51、纤维素结合模块（CBM）、Dockerin-X域（XDoc）和从鲁米诺球菌浮躁、烟草蚀刻病毒（TEV）蛋白酶、弹性蛋白样多肽（ELPs）的内聚力。 进行聚合酶链反应，并使用三限消化酶系统BamHI-BglII-KpnI对不同蛋白质片段的基因进行重组。<…

Representative Results

OaAEP1结扎在相邻蛋白质之间引入的NGL残留物不会影响聚合物中的蛋白质单体稳定性，因为展开力（<Fu>）和轮廓长度增量（<+Lc>;）与上一项研究（图1）相当。红霉素蛋白的纯化结果如图2所示。为了证明TEV裂解后的蛋白质与以下OaAEP1结扎相容，以构建具有控制序列的蛋白质聚合物，结构高效，图3提供了SDS-PAGE图像作…

Discussion

我们描述了一种酶生物合成和多蛋白固定化方案，并验证了基于AFM的SMFS的多蛋白设计。这种方法提供了一种新方法，以设计的顺序构建蛋白质聚合物，补充了以前多蛋白工程和固定4、6、52、53、54、55、56、57、58、59、60、61的方法。</su…

Divulgazioni

The authors have nothing to disclose.

Acknowledgements

这项工作得到了国家自然科学基金（授权号21771103，21977047），江苏省自然科学基金（授权号21771103）的支持。BK20160639）和江苏省双川计划。

Materials

iron (III) chloride hexahydrate Energy chemical 99%
Zinc chloride Alfa Aesar 100.00%
calcium chloride hydrate Alfa Aesar 99.9965% crystalline aggregate
L-Ascorbic Acid Sigma Life Science Bio Xtra, ≥99.0%, crystalline
(3-Aminopropyl) triethoxysilane Sigma-Aldrich ≥99%
sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate Thermo Scientific 90%
Glycerol Macklin 99%
5,5'-dithiobis(2-nitrobenzoic acid) Alfa Aesar
Genes Genscript
Equipment
Nanowizard 4 AFM JPK Germany
MLCT cantilever Bruker Corp
Mono Q 5/50 GL GE Healthcare
AKTA FPLC system GE Healthcare
Glass coverslip Sail Brand
Nanodrop 2000 Thermo Scientific
Avanti JXN-30 Centrifuge Beckman Coulter
Gel Image System Tanon
DOWNLOAD MATERIALS LIST

Riferimenti

  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. Yang, Y. J., Holmberg, A. L., Olsen, B. D. Artificially Engineered Protein Polymers. Annual Review of Chemical and Biomolecular Engineering. 8 (1), 549-575 (2017).
  3. Yang, J., et al. Polyprotein strategy for stoichiometric assembly of nitrogen fixation components for synthetic biology. Proceedings of the National Academy of Sciences of the United States of America. 115 (36), 8509-8517 (2018).
  4. Dietz, H., et al. Cysteine engineering of polyproteins for single-molecule force spectroscopy. Nature Protocols. 1 (1), 80-84 (2006).
  5. Carrion-Vazquez, M., et al. Mechanical and chemical unfolding of a single protein: A comparison. Proceedings of the National Academy of Sciences of the United States of America. 96 (7), 3694-3699 (1999).
  6. Hoffmann, T., et al. Rapid and Robust Polyprotein Production Facilitates Single-Molecule Mechanical Characterization of beta-Barrel Assembly Machinery Polypeptide Transport Associated Domains. ACS Nano. 9 (9), 8811-8821 (2015).
  7. Hoffmann, T., Dougan, L. Single molecule force spectroscopy using polyproteins. Chemical Society Reviews. 41 (14), 4781-4796 (2012).
  8. Deng, Y., et al. Enzymatic biosynthesis and immobilization of polyprotein verified at the single-molecule level. Nature Communications. 10 (1), 2775 (2019).
  9. Yuan, G., et al. Single-Molecule Force Spectroscopy Reveals that Iron-Ligand Bonds Modulate Proteins in Different Modes. The Journal of Physical Chemistry Letters. 10 (18), 5428-5433 (2019).
  10. Yang, R., et al. Engineering a Catalytically Efficient Recombinant Protein Ligase. Journal of the American Chemical Society. 139 (15), 5351-5358 (2017).
  11. Woodside, M. T., Block, S. M. Reconstructing Folding Energy Landscapes by Single-Molecule Force Spectroscopy. Annual Review of Biophysics. 43, 19-39 (2014).
  12. Sen Mojumdar, S., et al. Partially native intermediates mediate misfolding of SOD1 in single-molecule folding trajectories. Nature Communications. 8 (1), 1881 (2017).
  13. Singh, D., Ha, T. Understanding the Molecular Mechanisms of the CRISPR Toolbox Using Single Molecule Approaches. ACS Chemical Biology. 13 (3), 516-526 (2018).
  14. You, H., Le, S., Chen, H., Qin, L., Yan, J. Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers. Journal of Visualized Experiments. (127), e56328 (2017).
  15. Suren, T., et al. Single-molecule force spectroscopy reveals folding steps associated with hormone binding and activation of the glucocorticoid receptor. Proceedings of the National Academy of Sciences of the United States of America. 115 (46), 11688-11693 (2018).
  16. Tapia-Rojo, R., Eckels, E. C., Fernández, J. M. Ephemeral states in protein folding under force captured with a magnetic tweezers design. Proceedings of the National Academy of Sciences of the United States of America. 116 (16), 7873-7878 (2019).
  17. Chen, H., et al. Dynamics of Equilibrium Folding and Unfolding Transitions of Titin Immunoglobulin Domain under Constant Forces. Journal of the American Chemical Society. 137 (10), 3540-3546 (2015).
  18. Fu, L., Wang, H., Li, H. Harvesting Mechanical Work From Folding-Based Protein Engines: From Single-Molecule Mechanochemical Cycles to Macroscopic Devices. Chinese Chemical Society. 1 (1), 138-147 (2019).
  19. Scholl, Z. N., Li, Q., Josephs, E., Apostolidou, D., Marszalek, P. E. Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope. Journal of Visualized Experiments. (144), e55989 (2019).
  20. Zhang, S., et al. Towards Unveiling the Exact Molecular Structure of Amorphous Red Phosphorus by Single-Molecule Studies. Angewandte Chemie International Edition. 58 (6), 1659-1663 (2019).
  21. 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).
  22. Thoma, J., Sapra, K. T., Müller, D. J. Single-Molecule Force Spectroscopy of Transmembrane β-Barrel Proteins. Annual Review of Analytical Chemistry. 11 (1), 375-395 (2018).
  23. Chen, Y., Radford, S. E., Brockwell, D. J. Force-induced remodelling of proteins and their complexes. Current Opinion in Structural Biology. 30, 89-99 (2015).
  24. Takahashi, H., Rico, F., Chipot, C., Scheuring, S. alpha-Helix Unwinding as Force Buffer in Spectrins. ACS Nano. 12 (3), 2719-2727 (2018).
  25. Borgia, A., Williams, P. M., Clarke, J. Single-molecule studies of protein folding. Annu. Rev. Biochem. 77, 101-125 (2008).
  26. Florin, E., Moy, V., Gaub, H. Adhesion forces between individual ligand-receptor pairs. Science. 264 (5157), 415-417 (1994).
  27. Zakeri, B., et al. Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin. Proceedings of the National Academy of Sciences of the United States of America. 109 (12), 690-697 (2012).
  28. Ott, W., Jobst, M. A., Schoeler, C., Gaub, H. E., Nash, M. A. Single-molecule force spectroscopy on polyproteins and receptor-ligand complexes: The current toolbox. Journal of Structural Biology. 197 (1), 3-12 (2017).
  29. Stahl, S. W., et al. Single-molecule dissection of the high-affinity cohesin-dockerin complex. Proceedings of the National Academy of Sciences of the United States of America. 109 (50), 20431-20436 (2012).
  30. Oh, Y. J., et al. Ultra-Sensitive and Label-Free Probing of Binding Affinity Using Recognition Imaging. Nano Letters. 19 (1), 612-617 (2019).
  31. Vera Andrés, M., Carrion-Vazquez, M. Direct Identification of Protein-Protein Interactions by Single-Molecule Force Spectroscopy. Angewandte Chemie International Edition. 55 (45), 13970-13973 (2016).
  32. Yu, H., Heenan, P. R., Edwards, D. T., Uyetake, L., Perkins, T. T. Quantifying the Initial Unfolding of Bacteriorhodopsin Reveals Retinal Stabilization. Angewandte Chemie International Edition. 58 (6), 1710-1713 (2019).
  33. Jobst, M. A., Schoeler, C., Malinowska, K., Nash, M. A. Investigating Receptor-ligand Systems of the Cellulosome with AFM-based Single-molecule Force Spectroscopy. Journal of Visualized Experiments. (82), e50950 (2013).
  34. Stetter, F. W. S., Kienle, S., Krysiak, S., Hugel, T. Investigating Single Molecule Adhesion by Atomic Force Spectroscopy. Journal of Visualized Experiments. (96), e52456 (2015).
  35. Nadler, H., et al. Deciphering the Mechanical Properties of Type III Secretion System EspA Protein by Single Molecule Force Spectroscopy. Langmuir. , (2018).
  36. Giganti, D., Yan, K., Badilla, C. L., Fernandez, J. M., Alegre-Cebollada, J. Disulfide isomerization reactions in titin immunoglobulin domains enable a mode of protein elasticity. Nature Communications. 9 (1), 185 (2018).
  37. Huang, W., et al. Maleimide-thiol adducts stabilized through stretching. Nature Chemistry. 11 (4), 310-319 (2019).
  38. Li, Y. R., et al. Single-Molecule Mechanics of Catechol-Iron Coordination Bonds. ACS Biomaterials Science, Engineering. 3 (6), 979-989 (2017).
  39. Popa, I., et al. Nanomechanics of HaloTag Tethers. Journal of the American Chemical Society. 135 (34), 12762-12771 (2013).
  40. Xue, Y., Li, X., Li, H., Zhang, W. Quantifying thiol-gold interactions towards the efficient strength control. Nature Communications. 5, 4348 (2014).
  41. Wiita, A. P., Ainavarapu, S. R. K., Huang, H. H., Fernandez, J. M. Force-dependent chemical kinetics of disulfide bond reduction observed with single-molecule techniques. Proceedings of the National Academy of Sciences of the United States of America. 103 (19), 7222-7227 (2006).
  42. Pill, M. F., East, A. L. L., Marx, D., Beyer, M. K., Clausen-Schaumann, H. Mechanical Activation Drastically Accelerates Amide Bond Hydrolysis, Matching Enzyme Activity. Angewandte Chemie International Edition. 58 (29), 9787-9790 (2019).
  43. Conti, M., Falini, G., Samori, B. How strong is the coordination bond between a histidine tag and Ni-nitrilotriacetate? An experiment of mechanochemistry on single molecules. Angew. Chem. Int. Ed. 39 (1), 215-218 (2000).
  44. Beedle, A. E. M., Lezamiz, A., Stirnemann, G., Garcia-Manyes, S. The mechanochemistry of copper reports on the directionality of unfolding in model cupredoxin proteins. Nature Communications. 6, 7894 (2015).
  45. Li, H., Zheng, P. Single molecule force spectroscopy: a new tool for bioinorganic chemistry. Current Opinion in Chemical Biology. 43, 58-67 (2018).
  46. Zheng, P., Takayama, S. i. J., Mauk, A. G., Li, H. Hydrogen bond strength modulates the mechanical strength of ferric-thiolate bonds in rubredoxin. Journal of the American Chemical Society. 134 (9), 4124-4131 (2012).
  47. Lei, H., et al. Reversible Unfolding and Folding of the Metalloprotein Ferredoxin Revealed by Single-Molecule Atomic Force Microscopy. Journal of the American Chemical Society. 139 (4), 1538-1544 (2017).
  48. Yuan, G., et al. Multistep Protein Unfolding Scenarios from the Rupture of a Complex Metal Cluster Cd3S9. Scientific Reports. 9 (1), 10518 (2019).
  49. Zheng, P., Arantes, G. M., Field, M. J., Li, H. Force-induced chemical reactions on the metal centre in a single metalloprotein molecule. Nature Communications. 6, 7569 (2015).
  50. Arantes, G. M., Bhattacharjee, A., Field, M. J. Homolytic cleavage of Fe-S bonds in rubredoxin under mechanical stress. Angewandte Chemie International Edition. 52 (31), 8144-8146 (2013).
  51. Blake, P. R., et al. Determinants of protein hyperthermostability: purification and amino acid sequence of rubredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus and secondary structure of the zinc adduct by NMR. Biochimica. 30 (45), 10885-10895 (1991).
  52. Ott, W., Durner, E., Mediated Gaub, H. E. Enzyme-Mediated, Site-Specific Protein Coupling Strategies for Surface-Based Binding Assays. Angewandte Chemie International Edition. 57 (39), 12666-12669 (2018).
  53. Garg, S., Singaraju, G. S., Yengkhom, S., Rakshit, S. Tailored Polyproteins Using Sequential Staple and Cut. Bioconjugate Chemistry. 29 (5), 1714-1719 (2018).
  54. Veggiani, G., et al. Programmable Polyproteams Built Using Twin Peptide Superglues. Proceedings of the National Academy of Sciences of the United States of America. 113 (5), 1202-1207 (2016).
  55. Pelegri-O’Day, E. M., Maynard, H. D. Controlled Radical Polymerization as an Enabling Approach for the Next Generation of Protein-Polymer Conjugates. Accounts of Chemical Research. 49 (9), 1777-1785 (2016).
  56. Zheng, P., Cao, Y., Li, H. Facile method of constructing polyproteins for single-molecule force spectroscopy studies. Langmuir. 27 (10), 5713-5718 (2011).
  57. Zimmermann, J. L., Nicolaus, T., Neuert, G., Blank, K. Thiol-based, site-specific and covalent immobilization of biomolecules for single-molecule experiments. Nature Protocols. 5 (6), 975-985 (2010).
  58. Becke, T. D., et al. Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy. Journal of Visualized Experiments. (138), e58167 (2018).
  59. Liu, H. P., Ta, D. T., Nash, M. A. Mechanical polyprotein assembly using sfp and sortase-mediated domain oligomerization for single-molecule studies. Small Methods. 2 (6), (2018).
  60. Zhang, Y., Park, K. Y., Suazo, K. F., Distefano, M. D. Recent progress in enzymatic protein labelling techniques and their applications. Chemical Society Reviews. 47 (24), 9106-9136 (2018).
  61. Luo, Q., Hou, C., Bai, Y., Wang, R., Liu, J. Protein Assembly: Versatile Approaches to Construct Highly Ordered Nanostructures. Chemical Reviews. 116 (22), 13571-13632 (2016).
  62. Valle-Orero, J., Rivas-Pardo, J. A., Popa, I. Multidomain proteins under force. Nanotechnology. 28 (17), 174003 (2017).

Tags

OaAEP1Enzymatic SynthesisPolymerized ProteinSingle-molecule Force SpectroscopyProtein OligomerPolymerization DegreeProtein-based MaterialCysteinePotassium ChromateSulfuric AcidAPTESSulfo-SMCCGL-ELP-50 Cys-proteinCantilever Surface

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Citazione di questo articolo
Deng, Y., Zheng, B., Liu, Y., Shi, S., Nie, J., Wu, T., Zheng, P. OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy. J. Vis. Exp. (156), e60774, doi:10.3791/60774 (2020).
Scarica citazione
Ristampe e autorizzazioni

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image