1 research outputs found
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 inâ
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 photoâactivatable 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 photoâactivation using 365â
nm light, binding is restored. We use this approach to generate improved photocaged variants of two antiâGFP 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 inâ
vivo effects on proteinâprotein interactions