Departamento de Microbiología, Facultad de Ciencias Bioquímicas y Farmacéuticas, Suipacha 531, Universidad Nacional de Rosario, Argentine National Research Council (CONICET), Rosario, Argentina.
Argentine Institute of Mathematics (IAM), Buenos Aires; Chemistry Institute - INQUISUR (UNS), Bahia Blanca, National Research Council - (CONICET), Argentina.
J Mol Biol. 2019 Mar 1;431(5):1016-1024. doi: 10.1016/j.jmb.2019.01.025. Epub 2019 Jan 25.
To address the mechanism of thermosensing and its implications for molecular engineering, we previously deconstructed the functional components of the bacterial thermosensor DesK, a histidine kinase with a five-span transmembrane domain that detects temperature changes. The system was first simplified by building a sensor that consists of a single chimerical transmembrane segment that retained full sensing capacity. Genetic and biophysical analysis of this minimal sensor enabled the identification of three modular components named determinants of thermodetection (DOTs). Here we combine and tune the DOTs to determine their contribution to activity. A transmembrane zipper represents the master DOT that drives a reversible and activating dimerization through the formation of hydrogen bonds. Our findings provide the mechanism and insights to construct a synthetic transmembrane helix based on a poly-valine scaffold that harbors the DOTs and regulates the activity. The construct constitutes a modular switch that may be exploited in biotechnology and genetic circuitry.
为了解决热敏感应机制及其对分子工程的意义，我们之前对细菌热敏传感器 DesK 的功能组件进行了解构，DesK 是一种具有五跨膜域的组氨酸激酶，可检测温度变化。该系统首先通过构建一个由单个嵌合跨膜片段组成的传感器得到简化，该传感器保留了完整的感应能力。对这种最小传感器的遗传和生物物理分析使我们能够识别三个模块化组件，分别命名为热敏检测决定因素（DOTs）。在这里，我们将 DOTs 进行组合和调整，以确定它们对活性的贡献。一个跨膜拉链代表了主 DOT，它通过氢键的形成驱动可逆的激活二聚化。我们的研究结果提供了一种机制和见解，可以基于多缬氨酸支架构建一个基于合成跨膜螺旋，该支架包含 DOTs 并调节其活性。该构建构成了一个模块化开关，可在生物技术和遗传电路中得到利用。
