# An  unnatural  amino acid system for capturing protein-protein interactions in Toxoplasma gondii

> **NIH NIH R21** · UNIVERSITY OF CALIFORNIA LOS ANGELES · 2020 · $224,742

## Abstract

PROJECT SUMMARY
The Apicomplexa are a large phylum of intracellular pathogens that cause substantial disease in humans and
animals worldwide. Their ability to infect their hosts, survive in their intracellular niche, and cause disease is
strictly dependent on a series of unique organelles that are common to this group of pathogens. As most of the
protein constituents of these organelles are not present in their human hosts, they represent intense areas of
investigation in the search for new drug targets. Toxoplasma gondii has served as a model system for the
study of apicomplexan parasites due of its ease of genetic manipulation and extensive set of molecular tools
that have been developed for its study. While these molecular tools have focused on dissecting function at the
individual gene level, the next big advances will require new approaches that are better able to evaluate
precisely how proteins interact in complexes and how they participate in networks within the parasite to enable
infections. To develop new tools that function at the protein level, we have been developing Toxoplasma
strains with an expanded genetic code that can incorporate unnatural amino acids (UAAs) to study protein-
protein interactions in vivo. This strategy uses an amber stop codon suppression system that enables strains to
incorporate a photoreactive UAA at specific sites into a bait protein, which crosslink to binding partners when
activated by UV light. We have demonstrated that parasites engineered to express an orthogonal amber
suppressor tRNA and aminoacyl-tRNA synthetase pair efficiently incorporate the photoactivatable UAA p-
azidophenylalanine (Azi) into control proteins and have also shown that we can obtain robust photoreactive
crosslinking using this approach. In this application, we will engineer our system to enable incorporation of a
second photoreactive UAA, p-benzoylphenylalanine. We will then utilize these systems to determine the
precise interactions of the TgARO complex, which is critical for the function of the secretory rhoptries, as well
as the interactions of a newly discovered protein in the conoid, which is important for invasion. Thus, this
application develops new tools for protein-protein interaction studies in Toxoplasma and applies these tools to
the study of critical protein complexes in the parasite.

## Key facts

- **NIH application ID:** 9850542
- **Project number:** 5R21AI135661-02
- **Recipient organization:** UNIVERSITY OF CALIFORNIA LOS ANGELES
- **Principal Investigator:** Peter John Bradley
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $224,742
- **Award type:** 5
- **Project period:** 2019-01-15 → 2021-12-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9850542

## Citation

> US National Institutes of Health, RePORTER application 9850542, An  unnatural  amino acid system for capturing protein-protein interactions in Toxoplasma gondii (5R21AI135661-02). Retrieved via AI Analytics 2026-06-11 from https://api.ai-analytics.org/grant/nih/9850542. Licensed CC0.

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