Extended Data Fig. 5: Design, expression and characterisation of cysteine-locked PfRH5.
From: The PfRCR complex bridges malaria parasite and erythrocyte during invasion
a, PfRH5 in cartoon showing the location of the five designed disulphide bonds (CC1 to CC5, black sticks), joining the N-terminal (dark yellow) and C-terminal (light yellow) halves of PfRH5. b, Gel filtration traces of purified wild-type PfRH5ΔNL (a construct lacking the unstructured N-terminus and α2-α3 loop of PfRH53) and the cysteine-locked PfRH5 designs (CC1 to CC5) following expression in Drosophila S2 cells. c, SDS-PAGE gel of purified wild-type and single cysteine-locked variants (CC1 to CC5) and the double cysteine-locked (PfRH5CL, which combines crosslinks CC1 and CC5) version of PfRH5ΔNL. CC1 to CC5 were each expressed and purified once, while PfRH5CL is representative of 3 independent samples. d, Single circular dichroism traces of wild-type and cysteine-locked variants of PfRH5ΔNL at 20 °C from their respective temperature ramps. e, Relative ellipticity at 220 nm versus temperature, and the calculated melting temperature of wild-type and cysteine-locked variants of PfRH5ΔNL. f, Circular dichroism traces of purified wild-type and the double cysteine-locked PfRH5ΔNL (PfRH5ΔNLCL). g, Intact mass spectrometry analysis of PfRH5ΔNLCL following maleimide-PEG-biotin labelling in the absence (top) and the presence of TCEP (bottom). Unlabelled PfRH5ΔNLCL has a theoretical mass of 41211.6 Da and +526 Da mass differences correspond to a maleimide-PEG2-biotin addition to cysteine residues.
