Document Type

Thesis

Degree Name

Master of Science (MSc)

Department

Biology

Program Name/Specialization

Integrative Biology

Faculty/School

Faculty of Science

First Advisor

Matthew Smith

Advisor Role

Thesis Supervisor

Abstract

Protein-protein interactions are critical for many biochemical processes and for cellular function. Understanding interactions between proteins and ligands, as well as other proteins are necessary for the modelling of biological systems. Protein import into chloroplasts is one such system where the understanding of interactions between members of the translocation complex is limited. The translocon at the outer envelope membrane of chloroplasts (Toe complex) consists of three core components: Toc75, Tocl59, and Toc34. Members of the Tocl59 and Toc34 gene families in Arabidopsis thaliana have been shown to assemble differentially into structurally and functionally distinct complexes. More specifically, there is evidence that complexes containing atTocl59 and atToc33 (the prefix “at” identifies the species of origin, Arabidopsis thaliana) import photosynthetic proteins, whereas complexes containing atTocl32/120 and atToc34 seem to preferentially import non-photosynthetic proteins. The interactions between atTocl59 and atToc33 or atTocl32 and atToc34 are mediated by their GTPase domains. The current research used recombinant GTPase domains to investigate the specifics of the interaction between atTocl59G and atToc33G in more detail using molecular and biophysical approaches. Methods included using blue-native PAGE, fluorescence spectroscopy and circular dichroism spectroscopy to characterize the interaction between atTocl59G and atToc33G. Objectives were to characterize the interaction between atTocl59G and atToc33G as well as attempt to manipulate the monomendimer equilibrium for individual proteins for the purpose of studying heterodimer formation. A secondary objective was to generate single tryptophan mutants of atTocl59G (W973F and W1056F) to more precisely characterize the interaction between proteins. Results indicate that only a small percentage of the total protein exists as heterodimers, making quantification using fluorescence spectroscopy and circular dichroism spectroscopy difficult. Nonetheless, it was demonstrated that heterodimer formation occurs between the G-domains of atTocl59 and atToc33. The data also suggest that higher order oligomers may form, which may be indicative of multiple interaction domains among proteins as previously suggested by stoichiometric studies on isolated Toe complexes. The effects of chelating agents, such as EDTA and Chelex, produced significant structural changes which also inhibited homo-dimerization indicating the specific need for GTP-nucleotide to maintain a functional conformation. Single tryptophan mutants of atTocl59G also produced major structural changes to recombinant proteins highlighting the importance of these residues for overall structure of the protein.

Convocation Year

2009

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