PASTEUR, Département de Chimie, École Normale Supérieure, PSL University, Sorbonne Université, CNRS, 24, rue Lhomond, Paris 75005, France.
Laboratory for Nanobiology, Department of Chemistry, KU Leuven, Celestijnenlaan 200G, Heverlee 3001, Belgium.
ACS Sens. 2021 Mar 26;6(3):1157-1165. doi: 10.1021/acssensors.0c02414. Epub 2021 Feb 10.
Composed of a reversibly photoswitchable unit allosterically linked to a sensing module, reversibly photoswitchable sensors (rs-sensors) represent a new and attractive strategy to quantitatively read-out analyte concentrations. However, their kinetic response to illumination is complex, and much attention is required from the design to the application steps. Here, we exploit a generic kinetic model of rs-sensors which enables us to point to key thermokinetic parameters, such as dissociation constants and kinetic rates for exchange toward the analyte, and cross-sections for photoswitching. The application of the model allows to evaluate the robustness of the analyzed parameters and to introduce a methodology for their reliable use. Model and methodology have been experimentally tested on a newly reported calcium sensor based on a reversibly photoswitchable green fluorescent protein allosterically linked to a calcium-sensing module integrating calmodulin and an RS20 peptide.
由一个与感应模块连接的可反向光开关单元组成，可反向光开关传感器（rs-sensor）代表了一种新的、有吸引力的策略，可以定量读出分析物浓度。然而，它们对光照的动力学响应很复杂，从设计到应用步骤都需要高度关注。在这里，我们利用一个通用的 rs-sensor 动力学模型，使我们能够指出关键的热动力学参数，如解离常数和向分析物交换的动力学速率，以及光开关的截面。模型的应用允许评估分析参数的稳健性，并引入一种可靠使用它们的方法。该模型和方法已在一种新报道的钙传感器上进行了实验测试，该传感器基于一个可反向光开关的绿色荧光蛋白，与一个钙感应模块连接，该模块集成了钙调蛋白和一个 RS20 肽。
