Generation of Photocaged Nanobodies for Intracellular Applications in an Animal Using Genetic Code Expansion and Computationally Guided Protein Engineering**

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.

VHH Nanobody Versatility against Pentameric Ligand-Gated Ion Channels

“…Additionally, the Nbs themselves may also be light-activated through the insertion of a photoswitchable domain, such as the LOV2 of Avena sativa (A.s.-LOV2), 85 or through the incorporation of photocaged amino acids into the Nb sequence (Figure 14). 86 These Nbs could be used as tools to further study specific functional transitions of pLGICs, with high precision.…”

Regulation of IL-24/IL-20R2 complex formation using photocaged tyrosines and UV light

“…Third, due to the usually large interfacial areas that are stabilized by multiple non-covalent interactions (hydrogen bonds, hydrophobic interactions, etc. ), targeting PPI with single-residue perturbations is not straightforward, typically requiring extensive screening campaigns (Bridge et al, 2019;O'Shea et al, 2022). This contrasts with the photocontrol of interactions between proteins and small ligands or substrates, which typically depend on one or a reduced number of residues closely localized in space, e.g., an enzyme's catalytic site (Nguyen et al, 2014;Zhou et al, 2020).…”

“…First, NBY has been proposed as a universal proximal cage for the temporal blockage of protein activity (Wang et al, 2019;Wang et al, 2021). Second, NBY has been successfully employed to photocontrol antigen-antibody interactions (Bridge et al, 2019;Jedlitzke et al, 2019;Joest et al, 2021;Jedlitzke and Mootz, 2022;O'Shea et al, 2022;Yilmaz et al, 2022;Bridge et al, 2023).…”