# Measuring Protein-Protein Interactions using SPT-RESOLFT

> **NIH NIH R21** · UNIVERSITY OF NEW MEXICO · 2020 · $183,751

## Abstract

Project Summary
We will develop a method called SPT-RESOLFT that allows the efficient measurement of protein-protein
interaction (PPI) dissociation rates and stoichiometry of small complexes in live cells. The approach makes use
of Single Particle Tracking (SPT) and Reversible Saturable Optical Fluorescence Transitions (RESOLFT).
Fluorescence tags (labeling the proteins of interests) are activated within a small, ~ 100 nm diameter region.
The PPI lifetimes and stoichiometry are then inferred from the SPT trajectories as the small number of
activated proteins diffuse away from the activation spot. Stoichiometry is determined by change-point analysis
that quantifies single-step photobleaching. Dissociation rates are determined by the directly observed
divergence of two (or more) trajectories that were previously correlated in motion. In this project, we will
specifically target interactions between membrane proteins.
This method will speed up the data collection throughput by 10 to 100 times as compared to multi-color SPT
methods as well as provide stoichiometry information not available from tracking a sparse subset of proteins as
done with traditional SPT methods.
Our method will be capable of measuring both homo- and hetero-interactions. Homo-interactions will be
measured using a single-color fluorescent protein (FP) approach that takes advantage of the green-emitting
photo-switchable FP Dronpa. Hetero-interactions will use a two-color approach that combines Dronpa and the
red-emitting photo-activatable protein PAtagRFP. We will verify the method using positive and negative
controls with defined labeling stoichiometry as well as with the well-characterized Epidermal Growth Factor
Receptor (EGFR, ErbB1) system that changes dimerization kinetics upon stimulation with the EGF ligand.
Since dynamic molecular interactions are universally required for responses to signaling inputs, the new tools
generated will find application in many different signaling systems. The advantages of higher throughput and
increased labeling density will open new possibilities for the study of PPI under a variety of conditions,
including ligand dose responses and the influence of therapeutics.

## Key facts

- **NIH application ID:** 9938640
- **Project number:** 5R21GM132716-02
- **Recipient organization:** UNIVERSITY OF NEW MEXICO
- **Principal Investigator:** Keith A Lidke
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $183,751
- **Award type:** 5
- **Project period:** 2019-06-01 → 2023-05-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9938640

## Citation

> US National Institutes of Health, RePORTER application 9938640, Measuring Protein-Protein Interactions using SPT-RESOLFT (5R21GM132716-02). Retrieved via AI Analytics 2026-05-25 from https://api.ai-analytics.org/grant/nih/9938640. Licensed CC0.

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