Characterisation of structure and stability differences between the C-lobes of human and P. falciparum calmodulin in the presence of calmidazolium

Abstract: Malaria is a serious disease that can lead to fatal consequences if not treated. It is mainly spread via Plasmodium falciparum, a parasite carried by mosquitoes as host organisms. As a potential way of treating malaria, research is being done on possible inhibitors of calmodulin (CaM) in the parasite. CaM is a highly conserved protein found in all eukaryotes, and is important in many essential biochemical reactions. The potential inhibitor analysed in this study is calmidazolium (CZM). This study aims to characterise structure and stability differences between the C-lobes of human and P. falciparum CaM, while analysing the effect of the presence of CZM.  Previous studies have proven that CZM acts as an inhibitor to human CaM by binding to the C-lobe, with a dissociation constant in the nano molar range. In other studies, thermal stability measurements have shown that the secondary structure of P. falciparum CaM is more stable than that of human CaM.  In this study, the stability measurements showed that for the ANS binding site and around tyrosines, the C-lobe of human CaM was more stable than the C-lobe of P. falciparum CaM, knowledge which was previously unknown. When studying the entire secondary structure, the C-lobe of P. falciparum CaM was found to be more stable, which is in agreement with previous studies for the secondary structure of the complete CaM variants. For binding, the dissociation constants for both the C-lobe of human CaM and for the C-lobe of P. falciparum CaM were proven to be at a lower range than micro molar, most likely in the nano molar range. This is in agreement with earlier findings regarding the entire human CaM. Furthermore, CaM and CZM were proven to have their absorbance at the same wavelengths. Finally, several amino acid differences between the C-lobes of human and P. falciparum CaM were found that could play a role in binding and stability. One specific amino acid that was suggested to contribute to the stabilisation of the C-lobe of P. falciparum CaM was isoleucine. In the C-lobe of human CaM, these isoleucines were exchanged to threonine and arginine. Another amino acid difference that could potentially play a key role was the valine versus isoleucine, where valine might contribute to the stabilisation of the ANS binding site of the C-lobe of human CaM. To perform this study, the methods fluorescence spectroscopy, UV spectroscopy and circular dichroism were used, as well as several bioinformatic tools.  Overall, both stability and structure analyses have helped determine several differences between the two CaM variants, opening up possibilities to find an inhibitor that targets only the CaM of P. falciparum. CZM still remains as an interesting potential inhibitor, and can hopefully be a part of future research in malaria treatment.