Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Department of Chemistry, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA; Program in Molecular and Cellular Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Cell. 2021 Oct 28;184(22):5670-5685.e23. doi: 10.1016/j.cell.2021.09.026. Epub 2021 Oct 11.
We describe an approach to study the conformation of individual proteins during single particle tracking (SPT) in living cells. "Binder/tag" is based on incorporation of a 7-mer peptide (the tag) into a protein where its solvent exposure is controlled by protein conformation. Only upon exposure can the peptide specifically interact with a reporter protein (the binder). Thus, simple fluorescence localization reflects protein conformation. Through direct excitation of bright dyes, the trajectory and conformation of individual proteins can be followed. Simple protein engineering provides highly specific biosensors suitable for SPT and FRET. We describe tagSrc, tagFyn, tagSyk, tagFAK, and an orthogonal binder/tag pair. SPT showed slowly diffusing islands of activated Src within Src clusters and dynamics of activation in adhesions. Quantitative analysis and stochastic modeling revealed in vivo Src kinetics. The simplicity of binder/tag can provide access to diverse proteins.
我们描述了一种在活细胞中单粒子追踪（SPT）中研究单个蛋白质构象的方法。“结合物/标签”是基于将 7 肽（标签）掺入到其溶剂暴露由蛋白质构象控制的蛋白质中。只有在暴露时，肽才能与报告蛋白（结合物）特异性相互作用。因此，简单的荧光定位反映了蛋白质构象。通过直接激发明亮的染料，可以跟踪单个蛋白质的轨迹和构象。简单的蛋白质工程提供了非常适合 SPT 和 FRET 的高特异性生物传感器。我们描述了 tagSrc、tagFyn、tagSyk、tagFAK 和一个正交的结合物/标签对。SPT 显示在 Src 簇内激活的 Src 缓慢扩散的岛和粘着处激活的动力学。定量分析和随机建模揭示了 Src 的体内动力学。结合物/标签的简单性可以提供对不同蛋白质的访问。
