Browsing by Author "Shonhai, Addmore"

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    Comparative characterization of plasmodium falciparum Hsp70-1 relative to E. coli DnaK reveals the functional specificity of the parasite chaperone
    (Multidisciplinary Digital Publishing Institute (MDPI), 2020-06-04) Lebepe, Charity Mekgwa; Matambanadzo, Pearl Rutendo; Makhoba, Xolani Henry; Achilonu, Ikechukwu; Zininga, Tawanda; Shonhai, Addmore
    Hsp70 is a conserved molecular chaperone. How Hsp70 exhibits specialized functions across species remains to be understood. Plasmodium falciparum Hsp70-1 (PfHsp70-1) and Escherichia coli DnaK are cytosol localized molecular chaperones that are important for the survival of these two organisms. In the current study, we investigated comparative structure-function features of PfHsp70-1 relative to DnaK and a chimeric protein, KPf, constituted by the ATPase domain of DnaK and the substrate binding domain (SBD) of PfHsp70-1. Recombinant forms of the three Hsp70s exhibited similar secondary and tertiary structural folds. However, compared to DnaK, both KPf and PfHsp70-1 were more stable to heat stress and exhibited higher basal ATPase activity. In addition, PfHsp70-1 preferentially bound to asparagine rich peptide substrates, as opposed to DnaK. Recombinant P. falciparum adenosylmethionine decarboxylase (PfAdoMetDC) co-expressed in E. coli with either KPf or PfHsp70-1 was produced as a fully folded product. Co-expression of PfAdoMetDC with heterologous DnaK in E. coli did not promote folding of the former. However, a combination of supplementary GroEL plus DnaK improved folding of PfAdoMetDC. These findings demonstrated that the SBD of PfHsp70-1 regulates several functional features of the protein and that this molecular chaperone is tailored to facilitate folding of plasmodial proteins.
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    Supporting data on characterisation of linker switch mutants of Plasmodium falciparum heat shock protein 110 and canonical Hsp70
    (Elsevier Inc., 2021-05-29) Chakafana, Graham; Mudau, Pertunia T.; Zininga, Tawanda; Shonhai, Addmore
    Here, we present data on characterisation of the linker of Plasmodium falciparum Hsp110 (PfHsp70-z) relative to the linker of canonical Hsp70s in support of a co-published article [1]. The linker of PfHsp70-z was switched with that of canonical Hsp70s, represented by PfHsp70–1 (cytosolic counterpart of PfHsp70-z) and E. coli Hsp70/DnaK. The datasets represent comparative analyses of PfHsp70-z, PfHsp70–1, and E. coli DnaK, relative to their linker switch mutants; PfHsp70-zLS, PfHsp70–1LS, DnaKLS, respectively. Intrinsic and extrinsic fluorescence spectroscopic analyses were employed to elucidate effects of the mutations on the structural features of the proteins. The structural conformations of the proteins were analysed in the absence as well as presence of nucleotides. In addition, stability of the proteins to stress (pH changes and urea) was also determined. Surface plasmon resonance (SPR) was employed to determine affinity of the proteins for ATP. The relative affinities of PfHsp70-z and PfHsp70–1 for the parasite cytosol localised, J domain co-chaperone, PfHsp40, was determined by SPR analysis. The effect of the linker of PfHsp70-z on the interaction of DnaKLS with DnaJ (a co-chaperone of DnaK), was similarly determined. These data could be used for future investigations involving protein-protein/ligand interactions as described in [1]. The raw data obtained using the various techniques here described are hosted in the Mendeley Data repository at [2].