Allen K. Kim,corresponding author#1,2,3 Helen D. Wu,#1,2,3 and Takanari Inouecorresponding author1,2,3
1Department of Biomedical Engineering, School of Medicine, Johns Hopkins University, Baltimore, MD USA
2Department of Cell Biology, School of Medicine, Johns Hopkins University, Baltimore, MD USA
3Center for Cell Dynamics, School of Medicine, Johns Hopkins University, Baltimore, MD USA
Allen K. Kim, Email: [email protected]
Molecular switches that respond to a biochemical stimulus in cells have proven utility as a foundation for developing molecular sensors and actuators that could be used to address important biological questions. Developing a molecular switch unfortunately remains difficult as it requires elaborate coordination of sensing and actuation mechanisms built into a single molecule. Here, we rationally designed a molecular switch that changes its subcellular localization in response to an intended stimulus such as an activator of protein kinase A (PKA). By arranging the sequence for Kemptide in tandem, we designed a farnesylated peptide whose localization can dramatically change upon phosphorylation by PKA. After testing a different valence number of Kemptide as well as modulating the linker sequence connecting them, we identified an efficient peptide switch that exhibited dynamic translocation between plasma membranes and internal endomembranes in a PKA activity dependent manner. Due to the modular design and small size, our PKA switch can have versatile utility in future studies as a platform for visualizing and perturbing signal transduction pathways, as well as for performing synthetic operations in cells.
The sample was illuminated with a LED light source (pE-300, CoolLED, Andover, UK).
Product Associated Features
The pE-300white is a popular illuminator for everyday fluorescent screening and analysis with simple operatation and individual irradiance control of each LED channel.
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