PROJECT SUMMARY Nature has evolved dynamic proteins that act as sensors, detecting changes in the environment and producing signals that allow the cell to adapt and respond accordingly. For this function, signaling proteins must adopt multiple conformations, affording switch-like behavior between “on” and “off” states. The specific mechanisms by which states are switched or maintained often remain elusive due to a lack of biochemical and structural information. Herein, I propose that protein design and engineering offer a unique approach to deconvolute these complex biomolecular processes. Specifically, I aim to use protein design to generate minimal, tunable versions of modular signaling subdomains and engineer them into wild-type signaling protein scaffolds. This allows for direct assessment of thermodynamic or conformational signaling mechanisms by allowing us to measure the effect of design changes on signaling outputs. For this proposal, bacterial histidine kinases offer a promising test system due to 1) their highly modular scaffold that is amenable to chimera engineering and 2) the coiled-coil core through which signal transduction is propagated, as coiled-coils are highly designable. In my preliminary efforts, I have demonstrated for the first time that a histidine kinase can be re-wired with a de novo designed sensor domain, allowing signal transduction to be initiated from a de novo part. The proposed research expands upon this by applying protein sequence design to vary sensor domain stability, enabling insight into how sensor stability (and thus, the thermodynamic gap between sensor domain “off” and “on” states) affects signaling output (Aim 1). Moving down the histidine kinase scaffold, I then propose to use hyperstable de novo designed helical bundles to drive the geometry (conformation) of the catalytic domain (Aim 2). By systematically varying the geometry of the de novo bundle, I can force the kinase domain to adopt a related geometry. Then, by measuring kinase activity of the de novo/kinase chimeras, I aim to identify geometric parameters at which the kinase domain switches activity states. Finally, I propose to apply multi-state design to generate de novo linker domains that can adopt multiple conformations (Aim 3). Engineering of de novo/natural chimeras and experimental characterization of multi-state designed candidates will shed light on the sequence diversity that enables accommodation of multiple conformations. Together, these efforts will provide significant mechanistic insight into how histidine kinases undergo signal transduction while pushing the boundaries of function-guided protein design.