Design of allosteric light regulation in multi-enzyme complexes

利用光人工控制酶是蛋白质工程和合成生物学中一门迅速兴起并得到广泛应用的学科。虽然通过在活性部位掺入光响应元件来调节单体酶是相对简单的，并且已经多次被证明，但在多酶复合体中光调节变构信号是更具挑战性的。在过去的资助期间，我们通过展示咪唑甘油磷酸合成酶(ImGPS)和色氨酸合成酶(TS)双酶复合体中变构的有效光调节来实现这一目标。为此，我们使用了非天然氨基酸，特别是可逆光开关非天然氨基酸(LsUAA)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用光调节变构的通用性和效率来将我们的策略提高到下一个复杂水平。为了增加通用性，我们计划实现新型的lsUA，它基于AzoF以外的可逆光开关发色团。为此，我们打算合成三种含有芳基偶氮吡唑、半硫代吲哚和硫代硫酰亚胺的异硫脲，它们都表现出良好的光化学性质，与AzoF互补。下一步，我们希望进化出对这三种lsUAAs具有高亲和力的氨基酰-tRNA合成酶，这将有助于它们在异源基因表达过程中的整合。最后，我们计划在我们的模型酶ImGPS和TS中测试每个lsUAA对变构的光调节的潜力。我们特别感兴趣的是，这些lsUAAs是否表现出比AzoF更高的光调节因子，以及它们是否能够在AzoF没有作用的位置调节酶。为了提高效率，我们旨在通过定向进化来优化光响应型UAA的蛋白质环境。具体地说，我们计划扩大两个ImGPS复合体的光调节潜力，这两个复合体在不同的位置含有AzoF，它们都只表现出对活性和变构的适度调节。为此，我们将针对AzoF附近的位置进行半理性设计实验。通过使用光响应活动筛选，我们希望找到能够进一步将光调节因子提高至少一个数量级的变体。综上所述，该项目的结果将为创造具有无与伦比的性能的可逆光控制变构酶铺平道路。

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.