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

We describe an approach to study the conformation of individual proteins during single particle tracking (SPT) in living cells. Binder/tagis 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.

Automated summary

The approach utilizes binder/tag to study the conformation of individual proteins during single particle tracking in living cells, allowing for the tracking of protein trajectory and conformation, and providing highly specific biosensors.