相关实验视频

Updated: Jul 31, 2026

Glutamine Flux Imaging Using Genetically Encoded Sensors

Glutamine Flux Imaging Using Genetically Encoded Sensors

Published on: July 31, 2014

尼特罗夫:一种工程光生物传感器,用于照亮活细胞中的酸盐运输

Mariah A Cook¹, Jonathan D Smailys², Ke Ji¹

¹Department of Chemistry and Biochemistry, The University of Texas at Dallas, Richardson, TX 75080.

bioRxiv : the preprint server for biology

|April 1, 2025

概括

研究人员开发了一种新的光生物传感器NitrOFF,用于哺乳动物细胞中实时酸盐成像. 这种工具有助于人们更好地了解酸盐.

科学领域:

生物化学生物化学
分子生物学分子生物学
细胞生物学细胞生物学

背景情况:

酸盐在人类生理学中起着双重作用,既是污染物,又是重要的营养物质/信号离子.
对于酸盐生物学的基本理解,特别是吸收和储存,是有限的.
基因编码的光生物传感器为实时酸盐成像提供了有希望的方法,但哺乳动物细胞应用很少.

研究的目的:

设计和设计一种新的光生物传感器,用于检测哺乳动物细胞中的酸盐.
创建一个实时成像酸盐动态的工具.
克服现有的酸盐检测技术在细胞环境中的局限性.

主要方法:

通过将分裂的绿色光蛋白 (EGFP) 与酸盐识别域 (NreA) 融合,构建了一个两域的虚构体.
采用7轮定向进化,引入15个突变来开发功能生物传感器,命名为NitrOFF.
利用X射线晶体学来确定apo和酸盐结合的NitrOFF的结构,揭示了形状变化.

主要成果:

工程生物传感器NitrOFF表现出一个关闭强度度响应,解离常数 (Kd) 约为9μM.
在X射线结构中,在酸盐结合时,EGFP和NreA域之间显著的大规模形状重排 (68.4°变化).
这种重新排列是由链接器中螺旋体的形成驱动的,导致EGFP的β7链脱离并灭光.

结论:

NitrOFF是第一个功能性的光生物传感器,用于哺乳动物细胞中的酸盐,可实现实时监测.
生物传感器的机制涉及显著的全沟通和酸盐结合后的形状变化.
在人类胚胎脏 (HEK) 293细胞中监测外源酸盐吸收和调节的概念验证.

关键词:

在X射线晶体学.生物传感器生物传感器指导进化是指导进化的.绿色光蛋白是一种绿色光蛋白.酸盐酸盐的使用方法

更多相关视频

Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters CNiFERs for Optical Detection of Neurotransmitters In Vivo

Construction of Cell-based Neurotransmitter Fluorescent Engineered Reporters CNiFERs for Optical Detection of Neurotransmitters In Vivo

Published on: May 12, 2016

Application of Genetically Encoded Fluorescent Nitric Oxide (NO•) Probes, the geNOps, for Real-time Imaging of NO• Signals in Single Cells

Application of Genetically Encoded Fluorescent Nitric Oxide (NO•) Probes, the geNOps, for Real-time Imaging of NO• Signals in Single Cells

Published on: March 16, 2017

相关概念视频

Channel Rhodopsins

Channel Rhodopsins

Most organisms use photoreceptors to sense and respond to light. Examples of photoreceptors include bacteriorhodopsins and bacteriophytochromes in some bacteria, phytochromes in plants, and rhodopsins in the photoreceptor cells of the vertebral retina. The light-sensitive property of these receptors is because of the bound chromophores, such as bilin in the phytochromes and retinal in the rhodopsins.
Rhodopsins belong to the family of cell surface proteins called G-protein coupled receptors,...

G-Protein Gated Ion Channels

G-Protein Gated Ion Channels

GPCRs are primarily responsible for our sense of smell, taste, and vision. The binding of a sensory stimulus activates GPCR to stimulate effector proteins, many of which are ion channels in the sensory organs. GPCRs modulate the opening and closing of the target ion channels either directly by binding them, or by releasing second messengers that activate these channels. As ions move across the membrane, the membrane potential is altered, which induces an appropriate response.
Sensory organs,...

Microbial Biosensors

Microbial Biosensors

Microbial biosensors are analytical devices that utilize living microbes to detect specific substances through measurable signals. These devices consist of two main components: biosensing organisms and signal-transducing elements. Biosensing organisms, such as Escherichia coli or Saccharomyces cerevisiae, are typically housed in multiwell plates connected to transducers, enabling rapid, real-time detection of target analytes.Signal Generation MechanismWhen a target analyte—such as...