Generation of Photocaged Nanobodies for Intracellular Applications in an Animal Using Genetic Code Expansion and Computationally Guided Protein Engineering. (7th July 2022)

• Record Type: Journal Article
• Authors: O'Shea, Jack M.; Goutou, Angeliki; Brydon, Jack; Sethna, Cyrus R.; Wood, Christopher W.; Greiss, Sebastian
• Is Part Of: Chembiochem. Volume 23:Number 16(2022)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2022-07-07
• Subjects: C. elegans -- computational alanine scanning -- nanobodies -- photocaged non-canonical amino acids -- protein engineering; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pharmaceutical chemistry -- Periodicals; 572
• DOI: 10.1002/cbic.202200321
• Languages: English
• ISSNs: 1439-4227

Abstract

Abstract: 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. Abstract : Light‐activated nanobodies for intracellular use were created using a combination of genetic code expansion and computationally guided engineering of the binding interface. The engineered nanobodies allow optical control of subcellular protein localisation in living Caenorhabditis elegans .