Biochemical and biophysical characterization of PFF1010c, a Plasmodium falciparum heat shock protein

Abstract

Since time immemorial malaria remains a health concern despite the extensive interventions to eliminate the disease. Plasmodium falciparum, the agent for the most lethal form of the disease, survives insults imposed by the human host response and the adverse effects of antimalarial drugs, thereby building resistance against this intervention. Amongst other means, the parasite employs the heat shock protein (HSP) machinery in response to the imposed hostilities. Compared to other malaria parasites, P. falciparum expresses a large complement of an HSP family known as the J domain proteins (JDPs). Hsp70 is a prominent molecular chaperone whose chaperone function is coordinated by JDPs. While several JDPs have been characterized, the role of PFF1010c, a JDP of P. falciparum remains unknown. This study explored the structural and functional features of PFF1010c using in silico approaches (BLASTP, PSIPRED, CLUSTALW, MEGA11, SOPMA, CDD/SPARCLE ESPRIPT, Alphafold, GalaxyHomomer, ProtParam, PlasmoDB, and STRING). A plasmid construct expressing recombinant PFF1010c was expressed using E. coli XL1 Blue. The secondary structure of the protein was investigated using circular dichroism spectroscopy. PFF1010c thermostability was assessed by ANS probe based extrinsic fluorescence spectroscopy. The functional cooperation of PFF1010c with PfHsp70-1 was investigated by monitoring refolding of heat-denatured luciferase in vitro. PFF1010c appeared to assume a fold constituted by two domains: the SVN motif-containing J domain and the Cterminal domain. PFF1010c was found to be thermostable. Biochemical and in silico studies suggested that the protein self-associates through the C-terminal domain. Also, PFF1010c abrogated the refolding activity of PfHsp70-1. Although PFF1010c appeared to interact with PfHsp70-1, it wasincapable of stimulating PfHsp70-1 chaperone function. This suggests that PFF1010c may interact with PfHsp70-1 possibly to improve the holdase chaperone function of PfHsp70-1 as well as to conserve the energy economy of the parasite cell.