美国北卡罗来纳大学Klaus M. Hahn、Timothy C. Elston等研究人员合作通过基于肽暴露的生物传感器显示活细胞中的单分子构象。相关论文于2021年10月11日在线发表于国际学术期刊《细胞》。

研究人员报道了一种研究活细胞内单颗粒追踪（SPT）期间单个蛋白质构象的方法。"粘合剂/标签"是基于将一个7-mer的肽（标签）纳入一个蛋白质，其溶剂暴露由蛋白质的构象控制。只有在暴露后，肽才能与报告蛋白（结合剂）发生特异性的相互作用。因此，简单的荧光定位反映了蛋白质的构象。通过直接激发明亮的染料，可以跟踪单个蛋白质的运动轨迹和构象。简单的蛋白质工程提供了适用于SPT和FRET的高度特异性生物传感器。

研究人员描述了tagSrc、tagFyn、tagSyk、tagFAK，以及一个正交的粘合剂/标签对。SPT显示了在Src簇内缓慢扩散的活化Src岛和粘附中的活化动态。定量分析和随机建模揭示了体内Src的动力学。粘合剂/标签的简单性可以提供对不同蛋白质的研究。

附：英文原文

Title: Biosensors based on peptide exposure show single molecule conformations in live cells

Author: Bei Liu, Orrin J. Stone, Michael Pablo, J. Cody Herron, Ana T. Nogueira, Onur Dagliyan, Jonathan B. Grimm, Luke D. Lavis, Timothy C. Elston, Klaus M. Hahn

Issue&Volume: 2021-10-11

Abstract: We describe an approach to study the conformation of individual proteins during singleparticle tracking (SPT) in living cells. “Binder/tag” is based on incorporation ofa 7-mer peptide (the tag) into a protein where its solvent exposure is controlledby protein conformation. Only upon exposure can the peptide specifically interactwith a reporter protein (the binder). Thus, simple fluorescence localization reflectsprotein conformation. Through direct excitation of bright dyes, the trajectory andconformation of individual proteins can be followed. Simple protein engineering provideshighly specific biosensors suitable for SPT and FRET. We describe tagSrc, tagFyn,tagSyk, tagFAK, and an orthogonal binder/tag pair. SPT showed slowly diffusing islandsof activated Src within Src clusters and dynamics of activation in adhesions. Quantitativeanalysis and stochastic modeling revealed in vivo Src kinetics. The simplicity of binder/tag can provide access to diverse proteins.

DOI: 10.1016/j.cell.2021.09.026

Source: https://www.cell.com/cell/fulltext/S0092-8674(21)01109-0