Engineering and exploiting synthetic allostery of NanoLuc luciferase.
Engineering and exploiting synthetic allostery of NanoLuc luciferase.
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NanoLuc荧光素酶的工程化和开发合成变构。
DOI：
10.1038/s41467-022-28425-2
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发表时间：
2022-02-10
影响因子：
16.6
通讯作者：
Alexandrov K
中科院分区：
文献类型：
--
作者：
Guo Z;Parakra RD;Xiong Y;Johnston WA;Walden P;Edwardraja S;Moradi SV;Ungerer JPJ;Ai HW;Phillips JJ;Alexandrov K
Allostery使蛋白质能够相互转换不同的生化信号，形成复杂的代谢和信号网络。我们假设，蛋白质的环状排列增加了新的N-和C-末端与蛋白质的活性中心通过增加局部结构紊乱的功能耦合的可能性。为了测试这一点，我们构建了一个合成变构版本的环状排列的NanoLuc荧光素酶，可以通过配体诱导的分子内非共价环化来激活。该开关模块对结合结构域及其配体的结构具有耐受性，并且可用于创建蛋白质和小分子的生物传感器。所开发的生物传感器覆盖了一系列发射波长，显示灵敏度低至50 pM，动态范围高达16倍，并可以量化其在人体体液中的同源配体。我们应用氢交换动力学质谱分析时间分辨的结构变化，在开发的生物传感器和观察配体介导的折叠新创建的终端。蛋白质的变构调节允许生化信号的转导和相互转换。在这里，作者构建了一个变构版本的环状排列的NanoLuc，它可以被配体激活;他们用它来创建生物传感器，以量化人体体液中的同源配体。
Allostery enables proteins to interconvert different biochemical signals and form complex metabolic and signaling networks. We hypothesize that circular permutation of proteins increases the probability of functional coupling of new N- and C- termini with the protein’s active center through increased local structural disorder. To test this we construct a synthetically allosteric version of circular permutated NanoLuc luciferase that can be activated through ligand-induced intramolecular non-covalent cyclisation. This switch module is tolerant of the structure of binding domains and their ligands, and can be used to create biosensors of proteins and small molecules. The developed biosensors covers a range of emission wavelengths and displays sensitivity as low as 50pM and dynamic range as high as 16-fold and could quantify their cognate ligand in human fluids. We apply hydrogen exchange kinetic mass spectroscopy to analyze time resolved structural changes in the developed biosensors and observe ligand-mediated folding of newly created termini. Allosteric regulation of proteins allows transduction and interconversion of biochemical signals. Here the authors construct an allosteric version of circular permutated NanoLuc that can be activated by a ligand; they use this to create biosensors to quantify their cognate ligand in human fluids.
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影响因子：
14.8
作者：
Dolberg TB;Meger AT;Boucher JD;Corcoran WK;Schauer EE;Prybutok AN;Raman S;Leonard JN
通讯作者：
Leonard JN
影响因子：
3.2
作者：
Cascales, Laura;Craik, David J.
通讯作者：
Craik, David J.
影响因子：
3.4
作者：
Aksel, Tural;Barrick, Doug
通讯作者：
Barrick, Doug
影响因子：
16.6
作者：
Dagliyan, Onur;Krokhotin, Andrey;Dokholyan, Nikolay, V
通讯作者：
Dokholyan, Nikolay, V
影响因子：
14.8
作者：
Dagliyan, Onur;Dokholyan, Nikolay, V;Hahn, Klaus M.
通讯作者：
Hahn, Klaus M.