Psalmopeotoxin II-an anti-malarial cysteine knot peptide extracted from Psalmopeous cambridgei: chemical synthesis and characterizations of wild-type and disulfide mutantsThitawan Prasertdee
( M.Sc. )
The purpose of this research was to chemically synthesize and characterize Psalmopeotoxin II (pcFK2), an anti-malarial peptide first extracted from Psalmopoeus cambridgei. PcFK2 is a 28-residue peptide containing three disulfide bridges and belongs to the Inhibitor Cystine Knot (ICK) superfamily. The six cysteine residues of PcFK2 are conserved similar to other members of the ICK superfamily, suggesting their critical role for either folding or function. In this study, PcFK2 was synthesized using manual Fmoc solid phase peptide synthesis (SPPS). The three disulfide bonds of PcFK2 were constructed by two different approaches, regioselective and random oxidative approaches. The resulting disulfide bond patterns of PcFK2 from the two approaches were verified by the HLPC-MS analysis of intact peptides and by the disulfide bond mapping using tryptic digestion. Implications of the disulfide bonds on the biophysical and biological properties of PcFK2 were studied using three disulfide mutants in which a particular pair of cysteines was replaced with two isosteric serine residues. Structures and biophysical characteristics of PcFK2 and its disulfide mutants were studied using far-UV CD and fluorescence spectroscopy. Biological activities of all variants were evaluated using antiplasmodial assay against the KI multidrug- resistant strain of P. falciparum. The experimental results showed that chemical synthesis of PcFK2 could be accomplished by manual Fmoc SPPS, but the three disulfide bridges in PcFK2 could not be correctly synthesized by the random oxidative strategy. Structural and biophysical analyses revealed that PcFK2 and the three disulfide mutants had similar structure to the twisted ß-sheet. However, the studies of disulfide bond removal indicated that the two native disulfide bonds, Cys9-Cys22 and Cys18-Cys27, had higher impact than Cys2-Cys19 on the core packing of the twisted ß-sheet conformation. From the thermal unfolding studies monitored by far-UV CD, the resulting data showed that all variants were unfolded in a two-state fashion and were thermally unstable under 37°C. The thennalunfolding data also demonstrated that removal of the disulfide bond which connected mostly unstructured portion of the peptide would have the stronger impact on stability. The antiplasmodial activity data reflected that PcFK2 wild-type inhibited the growth of P.falciparum in vitro with an ICso of 2.73 ± 0.78 µM, while all the three mutants had very high IC50. Although the two native Cys9-Cys22 and Cys18-Cys27 under this study affected the thermal and structural stability of PcFK2, the native Cys2-Cysl9 had the most distinguishable impact on biological activity of PcFK2.
Anti-malarial peptide / Psalmopoeus cambridgeil / Psalmopeotoxin I and III cysteine knot family/ regioselective disulfide bond formation / biophysical characterization / antiplasmodial activity/ biophysical-biological activity relationship