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One-step asparaginyl endopeptidase (OaAEP1)-based protein immobilization for single-molecule force spectroscopy
Enzymatic protein ligation has become the most powerful and widely used method for high-precision atomic force microscopy single-molecule force spectroscopy (AFM-SMFS) study of protein mechanics. However, this methodology typically requires the functionalization of the glass surface with a correspon...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
RSC
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9533667/ https://www.ncbi.nlm.nih.gov/pubmed/36320890 http://dx.doi.org/10.1039/d2cb00135g |
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author | Ding, Xuan Wang, Ziyi Zheng, Bin Shi, Shengchao Deng, Yibing Yu, Hanyang Zheng, Peng |
author_facet | Ding, Xuan Wang, Ziyi Zheng, Bin Shi, Shengchao Deng, Yibing Yu, Hanyang Zheng, Peng |
author_sort | Ding, Xuan |
collection | PubMed |
description | Enzymatic protein ligation has become the most powerful and widely used method for high-precision atomic force microscopy single-molecule force spectroscopy (AFM-SMFS) study of protein mechanics. However, this methodology typically requires the functionalization of the glass surface with a corresponding peptide sequence/tag for enzymatic recognition and multiple steps are needed. Thus, it is time-consuming and a high level of experience is needed for reliable results. To solve this problem, we simplified the procedure using two strategies both based on asparaginyl endopeptidase (AEP). First, we designed a heterobifunctional peptide-based crosslinker, GL-peptide-propargylglycine, which links to an N(3)-functionalized surface via the click reaction. Then, the target protein with a C-terminal NGL sequence can be immobilized via the AEP-mediated ligation. Furthermore, we took advantage of the direct ligation between primary amino in a small molecule and protein with C-terminal NGL by AEP. Thus, the target protein can be immobilized on an amino-functionalized surface via AEP in one step. Both approaches were successfully applied to the AFM-SMFS study of eGFP, showing consistent single-molecule results. |
format | Online Article Text |
id | pubmed-9533667 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | RSC |
record_format | MEDLINE/PubMed |
spelling | pubmed-95336672022-10-31 One-step asparaginyl endopeptidase (OaAEP1)-based protein immobilization for single-molecule force spectroscopy Ding, Xuan Wang, Ziyi Zheng, Bin Shi, Shengchao Deng, Yibing Yu, Hanyang Zheng, Peng RSC Chem Biol Chemistry Enzymatic protein ligation has become the most powerful and widely used method for high-precision atomic force microscopy single-molecule force spectroscopy (AFM-SMFS) study of protein mechanics. However, this methodology typically requires the functionalization of the glass surface with a corresponding peptide sequence/tag for enzymatic recognition and multiple steps are needed. Thus, it is time-consuming and a high level of experience is needed for reliable results. To solve this problem, we simplified the procedure using two strategies both based on asparaginyl endopeptidase (AEP). First, we designed a heterobifunctional peptide-based crosslinker, GL-peptide-propargylglycine, which links to an N(3)-functionalized surface via the click reaction. Then, the target protein with a C-terminal NGL sequence can be immobilized via the AEP-mediated ligation. Furthermore, we took advantage of the direct ligation between primary amino in a small molecule and protein with C-terminal NGL by AEP. Thus, the target protein can be immobilized on an amino-functionalized surface via AEP in one step. Both approaches were successfully applied to the AFM-SMFS study of eGFP, showing consistent single-molecule results. RSC 2022-08-30 /pmc/articles/PMC9533667/ /pubmed/36320890 http://dx.doi.org/10.1039/d2cb00135g Text en This journal is © The Royal Society of Chemistry https://creativecommons.org/licenses/by-nc/3.0/ |
spellingShingle | Chemistry Ding, Xuan Wang, Ziyi Zheng, Bin Shi, Shengchao Deng, Yibing Yu, Hanyang Zheng, Peng One-step asparaginyl endopeptidase (OaAEP1)-based protein immobilization for single-molecule force spectroscopy |
title | One-step asparaginyl endopeptidase (OaAEP1)-based protein immobilization for single-molecule force spectroscopy |
title_full | One-step asparaginyl endopeptidase (OaAEP1)-based protein immobilization for single-molecule force spectroscopy |
title_fullStr | One-step asparaginyl endopeptidase (OaAEP1)-based protein immobilization for single-molecule force spectroscopy |
title_full_unstemmed | One-step asparaginyl endopeptidase (OaAEP1)-based protein immobilization for single-molecule force spectroscopy |
title_short | One-step asparaginyl endopeptidase (OaAEP1)-based protein immobilization for single-molecule force spectroscopy |
title_sort | one-step asparaginyl endopeptidase (oaaep1)-based protein immobilization for single-molecule force spectroscopy |
topic | Chemistry |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9533667/ https://www.ncbi.nlm.nih.gov/pubmed/36320890 http://dx.doi.org/10.1039/d2cb00135g |
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