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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...

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Autores principales: O'Shea, Jack M., Goutou, Angeliki, Brydon, Jack, Sethna, Cyrus R., Wood, Christopher W., Greiss, Sebastian
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2022
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.
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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
title_full Generation of Photocaged Nanobodies for Intracellular Applications in an Animal Using Genetic Code Expansion and Computationally Guided Protein Engineering
title_fullStr Generation of Photocaged Nanobodies for Intracellular Applications in an Animal Using Genetic Code Expansion and Computationally Guided Protein Engineering
title_full_unstemmed Generation of Photocaged Nanobodies for Intracellular Applications in an Animal Using Genetic Code Expansion and Computationally Guided Protein Engineering
title_short Generation of Photocaged Nanobodies for Intracellular Applications in an Animal Using Genetic Code Expansion and Computationally Guided Protein Engineering
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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