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Generation of Photocaged Nanobodies for Intracellular Applications in an Animal Using Genetic Code Expansion and Computationally Guided Protein Engineering
Nanobodies are becoming increasingly popular as tools for manipulating and visualising proteins in vivo. The ability to control nanobody/antigen interactions using light could provide precise spatiotemporal control over protein function. We develop a general approach to engineer photo‐activatable na...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
John Wiley and Sons Inc.
2022
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9542635/ https://www.ncbi.nlm.nih.gov/pubmed/35731601 http://dx.doi.org/10.1002/cbic.202200321 |
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author | O'Shea, Jack M. Goutou, Angeliki Brydon, Jack Sethna, Cyrus R. Wood, Christopher W. Greiss, Sebastian |
author_facet | O'Shea, Jack M. Goutou, Angeliki Brydon, Jack Sethna, Cyrus R. Wood, Christopher W. Greiss, Sebastian |
author_sort | O'Shea, Jack M. |
collection | PubMed |
description | Nanobodies are becoming increasingly popular as tools for manipulating and visualising proteins in vivo. The ability to control nanobody/antigen interactions using light could provide precise spatiotemporal control over protein function. We develop a general approach to engineer photo‐activatable nanobodies using photocaged amino acids that are introduced into the target binding interface by genetic code expansion. Guided by computational alanine scanning and molecular dynamics simulations, we tune nanobody/target binding affinity to eliminate binding before uncaging. Upon photo‐activation using 365 nm light, binding is restored. We use this approach to generate improved photocaged variants of two anti‐GFP nanobodies that function robustly when directly expressed in a complex intracellular environment together with their antigen. We apply them to control subcellular protein localisation in the nematode worm Caenorhabditis elegans. Our approach applies predictions derived from computational modelling directly in a living animal and demonstrates the importance of accounting for in vivo effects on protein‐protein interactions. |
format | Online Article Text |
id | pubmed-9542635 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | John Wiley and Sons Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-95426352022-10-14 Generation of Photocaged Nanobodies for Intracellular Applications in an Animal Using Genetic Code Expansion and Computationally Guided Protein Engineering O'Shea, Jack M. Goutou, Angeliki Brydon, Jack Sethna, Cyrus R. Wood, Christopher W. Greiss, Sebastian Chembiochem Research Articles Nanobodies are becoming increasingly popular as tools for manipulating and visualising proteins in vivo. The ability to control nanobody/antigen interactions using light could provide precise spatiotemporal control over protein function. We develop a general approach to engineer photo‐activatable nanobodies using photocaged amino acids that are introduced into the target binding interface by genetic code expansion. Guided by computational alanine scanning and molecular dynamics simulations, we tune nanobody/target binding affinity to eliminate binding before uncaging. Upon photo‐activation using 365 nm light, binding is restored. We use this approach to generate improved photocaged variants of two anti‐GFP nanobodies that function robustly when directly expressed in a complex intracellular environment together with their antigen. We apply them to control subcellular protein localisation in the nematode worm Caenorhabditis elegans. Our approach applies predictions derived from computational modelling directly in a living animal and demonstrates the importance of accounting for in vivo effects on protein‐protein interactions. John Wiley and Sons Inc. 2022-07-07 2022-08-17 /pmc/articles/PMC9542635/ /pubmed/35731601 http://dx.doi.org/10.1002/cbic.202200321 Text en © 2022 The Authors. ChemBioChem published by Wiley-VCH GmbH https://creativecommons.org/licenses/by/4.0/This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ (https://creativecommons.org/licenses/by/4.0/) License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Research Articles O'Shea, Jack M. Goutou, Angeliki Brydon, Jack Sethna, Cyrus R. Wood, Christopher W. Greiss, Sebastian Generation of Photocaged Nanobodies for Intracellular Applications in an Animal Using Genetic Code Expansion and Computationally Guided Protein Engineering |
title | Generation of Photocaged Nanobodies for Intracellular Applications in an Animal Using Genetic Code Expansion and Computationally Guided Protein Engineering
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title_full | Generation of Photocaged Nanobodies for Intracellular Applications in an Animal Using Genetic Code Expansion and Computationally Guided Protein Engineering
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title_fullStr | Generation of Photocaged Nanobodies for Intracellular Applications in an Animal Using Genetic Code Expansion and Computationally Guided Protein Engineering
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title_full_unstemmed | Generation of Photocaged Nanobodies for Intracellular Applications in an Animal Using Genetic Code Expansion and Computationally Guided Protein Engineering
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title_short | Generation of Photocaged Nanobodies for Intracellular Applications in an Animal Using Genetic Code Expansion and Computationally Guided Protein Engineering
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title_sort | generation of photocaged nanobodies for intracellular applications in an animal using genetic code expansion and computationally guided protein engineering |
topic | Research Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9542635/ https://www.ncbi.nlm.nih.gov/pubmed/35731601 http://dx.doi.org/10.1002/cbic.202200321 |
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