Design of allosteric light regulation in multi-enzyme complexes

光对酶的人工调控是蛋白质工程和合成生物学中一门新兴且应用广泛的学科。虽然通过在活性位点掺入光响应元件来调节单体酶相对简单，并且已经多次证明，但多酶复合物中变构信号的光调节更具挑战性。在过去的资助期间，我们已经达到了这一目标，证明了有效的光调节变构内的双酶复合物咪唑甘油磷酸合酶（ImGPS）和色氨酸合酶（TS）。为此，我们使用非天然氨基酸，特别是可逆光可切换的非天然氨基酸（IsUAA）苯丙氨酸-4 '-偶氮苯（AzoF）。在这些原理验证研究的基础上，我们现在打算通过增加光调节变构的多功能性和效率，将我们的策略提升到下一个复杂的水平。为了增加多功能性，我们计划实施基于可逆光开关发色团而不是AzoF的新型lsUAA。为了这个目的，我们打算合成三个lsUAA含有芳基偶氮吡唑，hemithiodiode，和fulgimide，其中每一个显示有利的光化学性质的互补AzoF。在下一步中，我们希望进化出对这三种lsUAA具有高亲和力的氨酰-tRNA合成酶，这将促进它们在异源基因表达期间的掺入。最后，我们计划在我们的模型酶ImGPS和TS中测试每个lsUAA用于变构的光调节的潜力。我们特别感兴趣的是，这些lsUAA是否显示出比AzoF更高的光调节因子，以及它们是否可以在AzoF没有显示效果的位置调节酶。为了提高效率，我们的目标是通过定向进化优化光响应UAA的蛋白质环境。具体来说，我们计划扩展两个ImGPS复合物的光调节潜力，在不同的位置含有AzoF，这两个复合物都只表现出适度的活性和变构调节。为此，我们将进行半理性设计实验，以AzoF附近的位置为目标。通过使用光响应活性筛选，我们期望找到进一步将光调节因子提高至少一个数量级的变体。总之，该项目的结果将为创造具有无与伦比特性的新型可逆光可控变构酶铺平道路。

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.