Merozoite surface protein 2 (MSP2), one of the most abundant proteins on the merozoite surface of Plasmodium falciparum, is recognized to be important for the parasite’s invasion into the host cell and is thus a promising malaria vaccine candidate. However, mediated mainly by its conserved N-terminal 25 residues (MSP21–25), MSP2 readily forms amyloid fibril-like aggregates under physiological conditions in vitro, which impairs its potential as a vaccine component. In addition, there is evidence that MSP2 exists in aggregated forms on the merozoite surface in vivo. To elucidate the aggregation mechanism of MSP21–25 and thereby understand the behavior of MSP2 in vivo and find ways to avoid the aggregation of relevant vaccine in vitro, we investigated the effects of agitation, pH, salts, 1-anilinonaphthalene-8-sulfonic acid (ANS), trimethylamine N-oxide dihydrate (TMAO), urea, and sub-micellar sodium dodecyl sulfate (SDS) on the aggregation kinetics of MSP21–25 using thioflavin T (ThT) fluorescence. The results showed that MSP21–25 aggregation was accelerated by agitation, while repressed by acidic pHs. The salts promoted the aggregation in an anion nature-dependent pattern. Hydrophobic surface-binding agent ANS and detergent urea repressed MSP21–25 aggregation, in contrast to hydrophobic interaction strengthener TMAO, which enhanced the aggregation. Notably, sub-micellar SDS, contrary to its micellar form, promoted MSP21–25 aggregation significantly. Our data indicated that hydrophobic interactions are the predominant driving force of the nucleation of MSP21–25 aggregation, while the elongation is controlled mainly by electrostatic interactions. A kinetic model of MSP21–25 aggregation and its implication were also discussed.

Merozoite surface protein 2 (MSP2), an abundant glycosylphosphatidylinositol-anchored protein on the surface of Plasmodium falciparum merozoites, is a promising malaria vaccine candidate. MSP2 is intrinsically disordered and forms amyloid-like fibrils in solution under physiological conditions. The 25 N-terminal residues (MSP21–25) play an important role in both fibril formation and membrane binding of the full-length protein. In this study, the fibril formation and solution structure of MSP21–25 in the membrane mimetic solvents sodium dodecyl sulfate (SDS), dodecylphosphocholine (DPC), and trifluoroethanol (TFE) have been investigated by transmission electronic microscopy, turbidity, thioflavin T fluorescence, circular dichroism (CD), and nuclear magnetic resonance (NMR) spectroscopy. Turbidity data showed that the aggregation of MSP21–25 was suppressed in the presence of membrane mimetic solvents. CD spectra indicated that helical structure in MSP21–25 was stabilized in SDS and DPC micelles and in high concentrations of TFE. The structure of MSP21–25 in 50% aqueous TFE, determined using NMR, showed that the peptide formed an amphipathic helix encompassing residues 10–24. Low concentrations of TFE favored partially folded helical conformations, as demonstrated by CD and NMR, and promoted MSP21–25 fibril formation. Our data suggest that partially folded helical conformations of the N-terminal region of MSP2 are on the pathway to amyloid fibril formation, while higher degrees of helical structure stabilized by high concentrations of TFE or membrane mimetics suppress self-association and thus inhibit fibril formation. The roles of the induced helical conformations in membrane interactions are also discussed.