STABILIZATION OF TYPE-I BETA-TURN CONFORMATIONS IN PEPTIDES CONTAINING THE NPNA-REPEAT MOTIF OF THE PLASMODIUM-FALCIPARUM CIRCUMSPOROZOITE PROTEIN BY SUBSTITUTING PROLINE FOR (S)-ALPHA-METHYLPROLINE

The immunologically dominant central portion of the circumsporozoite (CS) surface protein on the malaria parasite Plasmodium falciparum contains a large number of tandemly repeated NPNA tetrapeptide motifs. The preferred secondary structure of this repeat unit in aqueous solution has been investigated with the aid of the secondary structure-inducing amino acid (S)-alpha-methylproline (p(Me)). H-1-Nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy have been used to probe the structures of synthetic peptides containing one to three tetrapeptide NP(Me)NA units. The far-UV CD spectra of these peptides show more intense negative bands at 215 nm than do similar peptides based on the NPNA motif. This and the temperature dependence of the peptide amide chemical shifts, the pattern of NOE connectivities, and the magnitude of (3)J coupling constants, derived from one- and two-dimensional NMR spectra of Ac(NP(Me)NA)(3)-OH, provide strong evidence for stable turnlike structures. From NOE distance and dihedral angle restraints, structures consistent with the NMR parameters were calculated. These reveal a stable hydrogen-bonded type-I beta-turn conformation (most likely present at 70-80% population) within each NP(Me)NA motif, stabilized by the backbone C-alpha methylation. Side chain to backbone hydrogen bonds involving the side chain amide groups of both asparagine residues also appear to impart stabilization to the turn conformation. No regular repeating conformations were detected in the linker regions connecting each NP(Me)NA unit. Polyclonal antisera raised in rabbits against (NP(Me)NA)(3) recognized intact P. falciparum sporozoites in an immunofluorescence assay as efficiently as antisera raised against (NPNA)(3). This indicates that the type-I beta-turn detected in the P-Me-containing peptide is closely related to the immunologically dominant portion of the folded CS protein. An improved knowledge of the three-dimensional structure of this protein may be of value for the design of second-generation synthetic malaria vaccines.