# Unagi-based encoded fluorescent protein kinase sensors

> **NIH NIH R21** · UNIVERSITY OF DELAWARE · 2020 · $230,321

## Abstract

Fluorescent protein biosensors have broad applications as probes of biological function. Protein
phosphorylation and signal transduction cascades control cellular function, and often are changed in cancer to
permit uncontrolled cell growth and invasion. Encoded fluorescent probes of protein phosphorylation have
been applied to understand numerous kinases, but most have substantial limitations due to the small
fluorescence changes upon phosphorylation (typically 5-10% change in fluorescence) and the large size of the
constructs (typically greater than 60 kDa). We will develop a novel approach to the fluorescent detection of
protein kinase activity based on the design of a phosphorylation-dependent version of the proto-fluorescent
protein UnaG. UnaG fluorescence depends on the binding of the ubiquitous metabolite bilirubin. We will
reengineer UnaG so that it binds bilirubin poorly, and thus is non-fluorescent, when it is non-phosphorylated. In
contrast, upon phosphorylation, UnaG will bind bilirubin tightly and become fluorescent. Because the
fluorescence of UnaG is reversible, in contrast to the fluorescence of GFP and GFP derivatives, the reversible
binding of bilirubin can form the basis of phosphorylation-dependent fluorescence. The small size of UnaG
allows UnaG-based protein kinase sensors to be employed as encoded kinase-responsive protein tags,
allowing localization of the sensors onto proteins of interest to more effectively interrogate intracellular
signaling. This approach also is expected to result in much larger fluorescence changes upon phosphorylation
than existing FRET-based encoded sensors, with the small size allowing more rapid development of
fluorescent protein kinase biosensors. The basis of the design is that UnaG fluorescence derives from the rigid
binding of the proto-fluorescent metabolite bilirubin. Thus, mutations which reduce binding or increase
dynamics are expected to prevent or reduce fluorescence. The basis of the designs herein is the observation
that serine/threonine phosphorylation induces a large disorder-to-order transition. Residues from UnaG will be
changed to threonine or serine, with an expected reduction in UnaG fluorescence due to increased dynamics in
bilirubin binding. Specific phosphorylation will lead to an increase in order and the restoration of native UnaG
fluorescence. The optimized sites of phosphorylation-dependent fluorescence will be identified and applied to
incorporate recognition sequences for multiple protein kinases. The phosphorylation-dependent UnaG protein
will be applied to examine protein kinase activity of multiple protein kinases in solution and in cells. A
ratiometric UnaG-based protein kinase biosensor will also be developed. This work will provide a broad
platform for phosphorylation-dependent fluorescent detection of protein kinase activity.

## Key facts

- **NIH application ID:** 9870941
- **Project number:** 5R21GM131302-02
- **Recipient organization:** UNIVERSITY OF DELAWARE
- **Principal Investigator:** Neal J Zondlo
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $230,321
- **Award type:** 5
- **Project period:** 2019-03-01 → 2021-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9870941

## Citation

> US National Institutes of Health, RePORTER application 9870941, Unagi-based encoded fluorescent protein kinase sensors (5R21GM131302-02). Retrieved via AI Analytics 2026-05-23 from https://api.ai-analytics.org/grant/nih/9870941. Licensed CC0.

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