Abstract
The genetic encoding of three different azobenzene phenylalanines with different photochemical properties was achieved in human cells by using an engineered pyrrolysyl tRNA/tRNA synthetase pair. In order to demonstrate reversible light control of protein function, azobenzenes were site-specifically introduced into firefly luciferase. Computational strategies were applied to guide the selection of potential photoswitchable sites that lead to a reversibly controlled luciferase enzyme. In addition, the new azobenzene analogues provide enhanced thermal stability, high photoconversion, and responsiveness to visible light. These small-molecule photoswitches can reversibly photocontrol protein function with excellent spatiotemporal resolution, and preferred sites for incorporation can be computationally determined, thus providing a new tool for investigating biological processes.