Today, the Hahn and Elston Labs published a paper in the journal Cell. Lead authors were Bei Liu, PhD, Assistant Professor in Pharmacology, and former graduate students Orrin Stone (Pharmacology) and Michael Pablo (Chemistry). The article describes a new approach to visualize the changing conformations of individual molecules in living cells, affording insight into the organization and dynamics of kinase signaling.
- Generalizable biosensors based on changes in exposure of small peptides
- A bright signal enables detection of single molecule conformations in living cells
- Conformation is revealed by FRET or simply by localization of a fluorophore
- Quantitation of Src dynamics and activation in clusters and at adhesions
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.
Co-authors are Cody Herron, Ana T. Nogueira, and Onur Dagliyan from Pharmacology, Jonathan B. Grimm and Luke D. Lavis from Janelia Farm Research Institute. The research for this article was funded by the National Institutes of Health (NIH) and by the Howard Hughes Medical Institute (HHMI).
UPDATE: UNC Healthcare highlights this article in their News: https://news.unchealthcare.org/2021/10/powerful-technique-allows-scientists-to-study-how-proteins-change-shape-inside-cells/