The phylum Apicomplexa consists of obligate intracellular parasites, many of which are prevalent pathogens of medical and veterinary importance. Toxoplasma gondii is one of the most successful parasites in the world, estimated to infect one-third of the human population, and poses potentially life-threatening complications in immunocompromised individuals and during a primary infection of a developing fetus. T. gondii also serves as a model organism for other apicomplexans such as Plasmodium spp., the causative agent of malaria, due to its ease of culture, high rate of transformation, and a rich set of tools available for genetic manipulation.

A common structure found in this phylum is the inner membrane complex (IMC), which is made of both membrane and cytoskeletal components and lays underneath the plasma membrane to provide a framework for replication and motility. An intermediate filament layer confers rigidity and tensile strength to the cell and these filaments are believed to be composed of a family of proteins called the alveolins. However, the exact organization of the proteins that make up this network is unknown and traditional methods for studying protein interactions are inadequate due to the detergent-insoluble nature of the IMC cytoskeleton.

Here, I successfully implement an unnatural amino acid (UAA) system in T. gondii using the photoactivated crosslinking UAA p-azidophenylalanine to study the IMC and other macromolecular complexes. This technique takes advantage of an orthogonal amber stop codon suppressor tRNA and cognate mutant aminoacyl-tRNA synthetase pair. In this system, the endogenous translational machinery directly incorporates Azi into the primary structure of a protein of interest. Upon exposure to ultraviolet light, Azi forms a covalent crosslinking between bait and prey proteins, enabling precise identification of the binding partner and overcoming the limitations of other protein interaction methods. Site-specific incorporation of Azi also maps the binding interface on the bait protein, providing structural information of the interaction.

After confirming that the requisite amber suppressor tRNA and aminoacyl-tRNA synthetase function properly and Azi can be used to crosslink protein complexes in the parasite, I applied this system towards IMC-localizing protein 1 (ILP1), which is a critical IMC protein involved in cytoskeletal integrity. I determine that ILP1 binds to three alveolins and one other cytoskeletal IMC protein, which is the first demonstration of interaction between components of the IMC intermediate filament network. I also explore the application towards a key protein complex that anchors the rhoptries, the secretory organelle that apicomplexans use to actively invade their host cell. This site-specific photoactivated UAA crosslinking system is a completely new approach to studying important protein complexes in T. gondii and provides new insight into apicomplexan biology.