# Single-molecule enzymology of protein kinases

> **NIH NIH R21** · UNIVERSITY OF TEXAS AT AUSTIN · 2022 · $225,465

## Abstract

Project Summary / Abstract
Protein kinases are ubiquitous signaling enzymes that regulate nearly every aspect of cell behavior. Kinase
activity is disrupted or misregulated in countless human diseases including cancer, metabolic diseases, and
neurological and developmental disorders. Kinases are regulated by upstream signals, which tune kinase
enzymatic activity by means of an array of binding partners and post-translational modifications. Although in
vitro reconstitution can be used to link binding partners and modifications to their effects on kinase enzymatic
activity, an in vitro approach is time consuming, challenging to apply to multiple partners/modifications
simultaneously, and not applicable to in vivo settings. This limitation has prevented the field from
understanding how kinases integrate upstream signals to establish an appropriate level of activity.
The long-term goal of the proposed research is to understand the biochemical basis of signal integration by
protein kinases in vivo. To enable progress towards this goal, new experimental tools are needed because
existing tools are unable to resolve the molecular state of a kinase molecule (i.e., its complement of
modifications and binding partners) and simultaneously measure its activity. This project focuses on
developing a single-molecule enzymatic assay for protein kinase activity. By studying single molecules, the
applicants will overcome the limitations of ensemble averaging, which would otherwise preclude directly linking
kinase modifications and binding partners to changes in activity because of the heterogenous nature of protein
complexes in vivo. A single-molecule approach will also enable direct application to cellular protein kinases,
eliminating the need for in vitro reconstitution.
The central objective of this work is to gain new technical knowledge that will enable application of
fluorescence-based kinase activity reporters in single molecule assays. The applicants will explore three
different approaches for isolating single kinase molecules, measuring their activity and simultaneously
determining which binding partners and post-translational modifications are present (Aim 1). Approaches that
appear promising will then be tested on cell-derived kinase molecules to determine whether application of
single-molecule kinase assays in vivo is feasible (Aim 2). The work proposed in this application is significant
because it will establish the feasibility of a novel approach to measuring protein kinase activity, with the
potential to ultimately yield fundamental insights into cellular signal transduction. The proposed work is
innovative, in the applicant’s opinion, because it represents a fundamentally new paradigm for studying
signaling by protein kinases. By establishing new tools with the potential to answer fundamental questions
about signal integration by cellular protein kinases, this work will contribute to the advancement of basic
biomedical research.

## Key facts

- **NIH application ID:** 10353547
- **Project number:** 1R21GM144817-01
- **Recipient organization:** UNIVERSITY OF TEXAS AT AUSTIN
- **Principal Investigator:** Daniel J Dickinson
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $225,465
- **Award type:** 1
- **Project period:** 2022-03-01 → 2024-02-29

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10353547

## Citation

> US National Institutes of Health, RePORTER application 10353547, Single-molecule enzymology of protein kinases (1R21GM144817-01). Retrieved via AI Analytics 2026-05-26 from https://api.ai-analytics.org/grant/nih/10353547. Licensed CC0.

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