Bacterial histidine kinases (HKs) are essential transmembrane signaling proteins that detect environmental stimuli and drive transcriptional responses. Despite their importance, we lack detailed knowledge of how signals are detected and transmitted to produce specific outputs. Large-scale studies of HK function can reveal the allosteric networks that regulate their activity.

Histidine kinases, key components of two-component systems, sense extracellular signals and trigger phosphorylation cascades that regulate gene expression. These systems are vital for bacterial adaptation and pathogenicity, requiring precise tuning of specificity and sensitivity. Generating large-scale E.coli HK genotype-phenotype relationships will enable us to parse out allosteric networks that enable intramolecular signal transduction and identify determinants of signaling specificity and output, providing molecular insights into the complex mechanisms by which these organisms sense and adapt to their environment.

With this dataset, we hope to answer:

1. How do changes in individual subdomains (alone and in combination) alter overall signaling profiles?

2. Do receptors that sense multiple stimuli rely on the same allosteric signaling pathways?

3. Is the modularity of these receptors predictable and engineerable?

Mechanistic insight into these sequence-function relationships will inform our understanding of how Nature has tuned these signaling systems and will advance our understanding of how signals are propagated through multiple modular subdomains. This will in turn enable future design and engineering of precisely tuned signaling systems with user-defined specificity, a long-standing goal in protein design and synthetic biology.