# Revealing signal transduction mechanisms through protein design

> **NIH NIH K99** · UNIVERSITY OF CALIFORNIA, SAN FRANCISCO · 2024 · $118,904

## Abstract

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.

## Key facts

- **NIH application ID:** 10948722
- **Project number:** 1K99GM155611-01
- **Recipient organization:** UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
- **Principal Investigator:** Anna Katherine Hatstat
- **Activity code:** K99 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $118,904
- **Award type:** 1
- **Project period:** 2024-09-01 → 2026-08-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10948722

## Citation

> US National Institutes of Health, RePORTER application 10948722, Revealing signal transduction mechanisms through protein design (1K99GM155611-01). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10948722. Licensed CC0.

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