The artificial control of enzymes by light is a rapidly emerging and widely applicable discipline of protein engineering and synthetic biology. While the regulation of monomeric enzymes by the incorporation of photo-responsive elements at the active site is relatively straightforward and has been shown several times, the light-regulation of allosteric signaling in multi-enzyme complexes is much more challenging. Within the past funding period, we have reached this goal by demonstrating efficient light-regulation of allostery within the bi-enzyme complexes imidazoleglycerol phosphate synthase (ImGPS) and tryptophan synthase (TS). To this end, we used unnatural amino acids, in particular the reversibly light-switchable unnatural amino acid (lsUAA) phenylalanine-4'-azobenzene (AzoF). Building on these proof-of-principle studies, we now intend to bring our strategy to the next level of sophistication by increasing the versatility and the efficiency of regulating allostery by light.To increase versatility, we plan to implement novel lsUAAs that are based on reversibly light-switchable chromophores other than AzoF. For this purpose, we intend to synthesize three lsUAAs containing arylazopyrazole, hemithioindigo, and fulgimide, each of which displays favorable photochemical properties complementary to AzoF. In the next step, we wish to evolve aminoacyl-tRNA synthetases with high affinity to these three lsUAAs, which will facilitate their incorporation during heterologous gene expression. Finally, we plan to test the potential of each lsUAA for the light regulation of allostery in our model enzymes ImGPS and TS. We are specifically interested to find out whether these lsUAAs show higher light regulation factors than AzoF, and whether they can regulate the enzymes at positions in which AzoF showed no effect. To increase efficiency, we aim at optimizing the protein environment of photo-responsive UAAs by directed evolution. Specifically, we plan to extend the light-regulation potential of two ImGPS complexes, containing AzoF at distinct positions, which both exhibited only a modest regulation of activity and allostery. For this purpose, we will perform semi-rational design experiments targeting positions in the vicinity of AzoF. By using a light-responsive activity screening, we expect to find variants that further improve the photo- regulation factor by at least one order of magnitude. Taken together, the results of this project will pave the way for creating new reversibly photo-controllable allosteric enzymes with unrivaled properties.
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