Abstract Description

Trypanosoma brucei is a unicellular parasitic protozoan responsible African sleeping sickness a fatal yet neglected tropical disease affecting populations in sub-Saharan Africa. The parasite’s survival within the mammalian host and tsetse fly vector relies on its ability to adapt to drastic environmental changes, including oxidative and thermal stress. Among the molecular systems that enable this adaptation, the heat shock protein 70 (Hsp70) family plays a central role in maintaining protein homeostasis by assisting in protein folding, refolding, and protection against denaturation.TbHsp70.c, a cytosolic Hsp70 homologue in T. brucei, has been identified as an essential component of the parasite’s stress response machinery. Interestingly, this protein displays distinct structural characteristics compared to canonical Hsp70s found in other eukaryotes. Notably, TbHsp70.c lacks the conserved C-terminal EEVD motif, which in most Hsp70s serves as a critical recognition site for co-chaperones and partner proteins. The absence of this motif suggests that TbHsp70.c may rely on alternative structural elements or unique regulatory mechanisms to execute its chaperone functions. To investigate the structure–function relationship of TbHsp70.c, three site-directed variants were generated: TbHsp70.c-ARCH, TbHsp70.c-Linker, and TbHsp70.c-Lid. Each variant targets a region known to be functionally critical in canonical Hsp70 proteins. The ARCH region, located within the substrate-binding domain (SBD), is implicated in substrate recognition and binding, influencing how the chaperone interacts with unfolded client proteins, the residues mutated were arch residues D412A and V437Y and substrate binding residues V409L and Y434F. The linker region connects the nucleotide-binding domain (NBD) and the SBD, and serves as a key communication bridge that transmits conformational signals between these domains during the ATP hydrolysis cycle. The lid region, an α-helical subdomain within the SBD, acts as a dynamic flap that opens or closes over the peptide-binding pocket the residues mutated are H475R and T548E. In this study, the wild-type TbHsp70.c and its three engineered variants will be expressed, purified, and characterized to compare their biochemical, structural, and functional properties.