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Supramolecular protein glue to boost enzyme activity

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  • ReceivedFeb 26, 2019
  • AcceptedMar 26, 2019
  • PublishedApr 17, 2019

Abstract

Proteins possess many biological functions. However, they can easily degrade or aggregate, thus losing their bioactivity. Therefore, it is very important to develop materials capable of interacting with proteins and forming nanostructures for protein storage and delivery. In this study, we serendipitously found a novel peptide-based supramolecular protein glue (Nap-GFFYK(γE)2-NH2, compound 1) that could co-assemble with proteins into nanofibers and hydrogels. We found that compound 1 rapidly folded into a β-sheet conformation upon contact with many proteins but not with polymers. Total internal reflection fluorescence microscopy (TIRFM) images clearly show the formation of co-assembled nanofibers by proteins and the peptide. The supramolecular protein glue could improve the dispersion of enzymes (lipase and lysozyme) and therefore enhance their catalytic activity, especially at high temperatures. More importantly, the supramolecular protein glue could co-assemble with two enzymes, glucose oxidase/horseradish peroxidase (GOx/HRP) and GOx/cytochrome c (cyt c), to form nanofibers that significantly enhanced the catalytic activity of tandem enzymatic reactions. We envisioned the great potential of our supramolecular protein glue for protein storage, delivery, and bioactivity manipulation.


Acknowledgment

This work was supported by the National Science Fund for Distinguished Young Scholars (31825012), the National Key Research and Development Program of China (2017YFC1103502), the National Natural Science Foundation of China (NSFC, 51773097, 51873156 and 21876116), Tianjin Science Fund for Distinguished Young Scholars (17JCJQJC44900), the National Program for Support of Top-notch Young Professionals, the Fundamental Research Funds for the Central Universities, and the Young Elite Scientists Sponsorship Program by Tianjin (TJSQNTJ-2017-16).


Interest statement

The authors declare no conflict of interest.


Contributions statement

Yang Z, Wang Q and Gao J designed the project and wrote the manuscript. Shang Y and Wang Z did the synthesis and the tests of TEM, CD, Rheology and MST. Liao Y performed enzyme activity tests. Ye Z and Xiao L did TIRFM. All authors helped with data analysis and manuscript preparation.


Author information

Yuna Shang received her BSc from Hebei University in 2016. Then she continued her study as a PhD candidate in Prof. Zhimou Yang’s Lab in Nankai University. Her research interest mainly focuses on the growth factors mimic peptides and biohybrid hydrogels based on proteins and peptides.


Jie Gao obtained a BSc degree in materials science and engineering from Tianjin University in 2008, and a PhD degree in polymeric chemistry and physics from Nankai University in 2013. Then she joined the Faculty of Nankai University in 2013, and now she is an associate professor of biomaterials. Her research interest focuses on the development of novel supramolecular hydrogels for biomedical applications.


Qigang Wang received his BSc and MSc from the East China University of Science and Technology in 1999 and 2002, respectively. He obtained his PhD degree in 2005 from Shanghai Institute of Ceramics, CAS under the supervision of Prof. Qiuming Gao. Before starting his independent research at Tongji University in March 2011, he was a postdoctoral fellow with Prof. Takuzo Aida at Tokyo University and Prof. Bing Xu at Hong Kong University of Science and Technology. His research interests focus on the mild bio-oxidative preparation and biomedical application of enzyme-laden hybrid hydrogel.


Zhimou Yang received his BSc from Nanjing University in 2001. He obtained his PhD degree in 2006 from Hong Kong University of Science and Technology under the supervision of Prof. Bing Xu. Before starting his independent research at Nankai University in March 2009, he was a postdoctoral fellow with Prof. Matthew Bogyo at Stanford Medical School. His research interests focus on molecular hydrogels of therapeutic agents (especially anti-cancer drugs) and short peptides and hydrogels based on protein-peptide interactions.


Supplement

Supplementary information

Supporting data are available in the online version of the paper.


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  • Figure 1

    (a) The chemical structure of 1. (b) Optical images of a solution of 1 (0.5 wt%) and the corresponding gel formed by adding GGT (13.8 U mL−1) immediately. (c) Schematic illustration for the formation of nanofibers of 1 and proteins.

  • Figure 2

    (a–f) TEM images of gels formed by adding lysozyme, trypsin, ACE, proteinase K, urease, and lipase (final concentration: 0.05 wt%), respectively, to solution of 1 (final concentration: 0.5 wt%) (scale bar: 100 nm). (g–i) TIRFM images of the nanofibers in the gel containing 0.5 wt% of 1 and 0.05 wt% of lipase.

  • Figure 3

    (a) CD spectra of hydrogels containing 0.5 wt% of 1 and 0.05 wt% of different proteins. (b) CD spectra of the solution of 1 at different time points after adding lipase. (c) The fitting curve of microscale thermophoresis (MST) to calculate the KD value between 1 or 2 and lipase. (d) The KD value of 1 with different proteins.

  • Figure 4

    Biocatalytic reaction courses (solid scatters) of different enzyme systems encapsulated in nanofiber and in free form fitted to linear fitting curves (solid lines). Hydrogel contains 1 wt% of 1 and (a) 0.1 wt% of lysozyme, (b) 0.1 wt% of lipase, (c) 0.1 wt% of GOx and 0.1 wt% of HRP, (d) 0.1 wt% of GOx and 0.1 wt% of cyt c. Free enzymes in PBS solutions are with the same content. (e–f) The biocatalytic activity of lipase and GOx/HRP cascade enzyme bounded in and without nanofiber at different temperatures.

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