Nanoscale probing and visualization of cyclic nucleotide-gated ion channels structural dynamics and function

环核苷酸门控离子通道结构动力学和功能的纳米级探测和可视化

• 批准号: 20K22626
• 负责人: Marchesi Arin
• 金额: $ 1.83万
• 依托单位: Kanazawa University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Research Activity Start-up
• 财政年份: 2020
• 资助国家: 日本
• 起止时间: 2020-09-11 至 2022-03-31
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-20K22626/
• 关键词: ion channels; gating; atomic force microscopy; membrane proteins; CNG channels; Atomic Force Microscopy; Ion channels; Purification; Gating

The SthK channel gene (UniProtKB G0GA88)was subcloned in the appropriate vector (pCGFP-BC) for overexpression in bacteria (BL21-DE3). Channel expression, purification and reconstitution in artificial bilayer was implemented successfully. The sample was characterized by means of force-spectroscopy (i.e. adsorbed to the AFM probe and unfolded from the membrane bilayer) and high-speed AFM imaging both in the presence of the endogenous ligand (cAMP) and its absence. This allowed us to define key interactions among the different protein domains and the overall mechanisms of gating (i.e. the opening of the permeation pathway). One paper, in collaboration with the ISAS-Italy (International School for Advanced Studies) is currently in revision and another is in preparation.

将SthK通道基因（UniProtKB G 0 GA88）亚克隆到适当的载体（pCGFP-BC）中，用于在细菌（BL 21-DE 3）中过表达。成功实现了通道在人工双层膜中的表达、纯化和重组。样品的特征在于通过力谱（即吸附到AFM探针和展开的膜双层）和高速AFM成像的内源性配体（cAMP）的存在和它的情况下，通过。这使我们能够定义不同蛋白质结构域之间的关键相互作用和门控的整体机制（即渗透途径的打开）。与意大利国际高级研究学院合作编写的一份文件目前正在修订，另一份正在编写。