Reversible and Tunable Photoswitching of Protein Function through Genetic Encoding of Azobenzene Amino Acids in Mammalian Cells

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.