Structural and functional studies on Trypanosoma brucei TTAGGG repeat-binding factor (tbTRF)

Objective The Telomere Repeat Binding Factor (TRF) in Trypanosoma brucei (tb) is the first identified telomere-associated protein in this flagellated protozoan parasite. It is essential for maintaining telomere architecture and cell viability. Furthermore it is implicated in the regulation of monoallelic expression of Variant Surface Glycoprotein (VSG), an antigenic variation mechanism employed by the parasite to evade host immune response. tbTRF contains two functional domains based on its homology to human TRFs, the Myb domain responsible for binding to double-stranded telomeric repeats and the homodimerization TRFH domain. Currently there is no structural information available regarding tbTRF, and its distant evolutionary relationship to higher-order eukaryotes renders it unreliable to apply the findings from human TRFs directly to tbTRF, particularly the role of tbTRF in VSG regulation has no counterpart in human system.;Methods In this thesis, we aim to carry out structural and biochemical characterization of the two functional domains in tbTRF, to understand its function in the telomere architecture and VSG regulation in T. brucei. Standard approaches were used to clone, express, and purify various constructs of tbMyb and tbTRFH domains. NMR was tried for structural studies. Biophysical and biochemical characterizations were carried out to assess protein stability and protein-DNA interactions for tbMyb domain.;Results We determined the tbMyb structure by NMR method. This domain structure is generally similar to the Myb domain of human TRFs. The interaction of tbMyb and telomeric DNA repeats were analyzed by NMR chemical shifts upon DNA titration and in vitro EMSA and ITC binding assays to identify the key residues for DNA binding. Mutants with abolished or weakened DNA-binding affinities were constructed based on structural findings, which were used to assess the impact of tbMyb domain on VSG regulation through in vivo studies, showing tbMyb domain may be involved in VSG switching rate regulation. Preliminary data has been obtained on the tbTRFH domain, demonstrating that it potentially forms a dimer. Our results can provide better understanding of the telomere biology in T. brucei and provide potential new insight into VSG-related anti-parasite drug design.