AI Article Synopsis
- The study introduces a method to observe individual protein conformations in live cells using a "binder/tag" approach, which relies on a peptide that interacts with a reporter protein only when the protein's conformation allows for exposure.
- This method enables researchers to track the movement and conformation of proteins through fluorescence, using engineered biosensors for precise monitoring.
- The findings report that activated Src proteins form slowly moving clusters within the cell, and the research highlights the potential for this technique to be applied to various proteins in biological studies.
Article Abstract
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.
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| http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8556369 | PMC |
| http://dx.doi.org/10.1016/j.cell.2021.09.026 | DOI Listing |
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