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

Abstract

The genetic encoding of three different azobenzene phenylalanines with different photochemical properties was achieved in human cells 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 selection of potential photoswitchable sites that lead to a reversibly controlled luciferase enzyme. Direct reversible photoswitching of enzymatic function in live cells has been accomplished through genetically encoded photoswitchable amino acids. In addition, the new azobenzene analogs 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.

Expanding the enzyme universe with genetically encoded unnatural amino acids

“…Reversible control over enzyme activity might be achieved by incorporation of photoisomerizable ncAAs, for example containing azobenzene side chains. 62,63 While azobenzene has been shown to allow for control over protein function, 64,65 no control over catalysis has been demonstrated yet with genetically encoded azobenzenes.…”

Modulating Protein–Protein Interactions with Visible‐Light‐Responsive Peptide Backbone Photoswitches

“…In particular, the group of Hecht has optimized the properties of classical azobenzenes by introducing σ‐electron‐withdrawing fluorine atoms in these positions, which leads to visible‐light switches with high photoconversions and very long lived cis isomers . Since this contribution, tetra‐ ortho ‐fluoroazobenzenes have been mainly used in materials science, and their applications in a biological context are scarce . Along these lines, there have been only a few cAzo derivatives used to modulate DNA hybridization and to control the helical conformation of peptides …”

Light-Regulation of Tryptophan Synthase by Combining Protein Design and Enzymology

“…Furthermore, reversible light-regulation is desirable. Light-switchable UAAs already exist [64,65,66], but in most studies, only light-regulation factors of 1.5–3 were achieved. Nevertheless, in our most recent study, we were able to obtain a light-regulation factor of ~10 by controlling the allosteric stimulation in the bi-enzyme complex imidazole glycerol phosphate synthase with the light-switchable UAA phenylalanine-4′-azobenzene (AzoF) [61].…”