Rational design and implementation of a chemically inducible heterotrimerization system.

Chemically inducible dimerization (CID) uses a small molecule to induce binding of two different proteins. CID tools such as the FK506-binding protein-FKBP-rapamycin-binding- (FKBP-FRB)-rapamycin system have been widely used to probe molecular events inside and outside cells. While various CID tools are available, chemically inducible trimerization (CIT) does not exist, due to inherent challenges in designing a chemical that simultaneously binds three proteins with high affinity and specificity. Here, we developed CIT by rationally splitting FRB and FKBP. Cellular and structural datasets showed efficient trimerization of split pairs of FRB or FKBP with full-length FKBP or FRB, respectively, by rapamycin. CIT rapidly induced tri-organellar junctions and perturbed intended membrane lipids exclusively at select membrane contact sites. By conferring one additional condition to what is achievable with CID, CIT expands the types of manipulation in single live cells to address cell biology questions otherwise intractable and engineer cell functions for future synthetic biology applications.

Synthetic design of farnesyl-electrostatic peptides for development of a protein kinase A membrane translocation switch.

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.

Rational Design of a Protein Kinase A Nuclear-cytosol Translocation Reporter.

Protein Kinase A (PKA) exists as a tetrameric holoenzyme which activates with increase of cAMP and plays an important role in many physiological processes including cardiac physiology, neuronal development, and adipocyte function. Although this kinase has been the subject of numerous biosensor designs, a single-fluorophore reporter that performs comparably to Förster resonance energy transfer (FRET) has not yet been reported. Here, we have used basic observations of electrostatic interactions in PKA substrate recognition mechanism and nucleus localization sequence motif to design a phosphorylation switch that shuttles between the cytosol and the nucleus, a strategy that should be generalizable to all basophilic kinases. The resulting reporter yielded comparable kinetics and dynamic range to the PKA FRET reporter, AKAR3EV. We also performed basic characterization and demonstrated its potential use in monitoring multiple signaling molecules inside cells using basic fluorescence microscopy. Due to the single-fluorophore nature of this reporter, we envision that this could find broad applications in studies involving single cell analysis of PKA activity.