Raman Srivatsan, Taylor Noah, Genuth Naomi, Fields Stanley, Church George M
Wyss Institute for Biologically-Inspired Engineering, Harvard University, 3 Blackfan Circle, Boston, MA 02115, USA; Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
Wyss Institute for Biologically-Inspired Engineering, Harvard University, 3 Blackfan Circle, Boston, MA 02115, USA; Department of Genetics, Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
Trends Genet. 2014 Dec;30(12):521-8. doi: 10.1016/j.tig.2014.09.004. Epub 2014 Oct 8.
Allosteric proteins have great potential in synthetic biology, but our limited understanding of the molecular underpinnings of allostery has hindered the development of designer molecules, including transcription factors with new DNA-binding or ligand-binding specificities that respond appropriately to inducers. Such allosteric proteins could function as novel switches in complex circuits, metabolite sensors, or as orthogonal regulators for independent, inducible control of multiple genes. Advances in DNA synthesis and next-generation sequencing technologies have enabled the assessment of millions of mutants in a single experiment, providing new opportunities to study allostery. Using the classic LacI protein as an example, we describe a genetic selection system using a bidirectional reporter to capture mutants in both allosteric states, allowing the positions most crucial for allostery to be identified. This approach is not limited to bacterial transcription factors, and could reveal new mechanistic insights and facilitate engineering of other major classes of allosteric proteins such as nuclear receptors, two-component systems, G protein-coupled receptors, and protein kinases.
变构蛋白在合成生物学中具有巨大潜力，但我们对变构分子基础的有限理解阻碍了设计分子的开发，包括具有新的DNA结合或配体结合特异性且能对诱导剂做出适当反应的转录因子。此类变构蛋白可作为复杂回路中的新型开关、代谢物传感器，或作为用于独立、可诱导控制多个基因的正交调节器。DNA合成和下一代测序技术的进步使得在单个实验中评估数百万个突变体成为可能，为研究变构提供了新机会。以经典的LacI蛋白为例，我们描述了一种使用双向报告基因的遗传筛选系统，以捕获处于两种变构状态的突变体，从而确定对变构最为关键的位置。这种方法不仅限于细菌转录因子，还可以揭示新的机制见解，并促进对其他主要类别的变构蛋白（如核受体、双组分系统、G蛋白偶联受体和蛋白激酶）的工程改造。
