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We investigated the immunogenicity and the conformational properties of the non-repetitive sequences of the Plasmodium falciparum circumsporozoite (CS) protein. Two polypeptides of 104 and 102 amino acids long, covering, respectively, the N- and C-terminal regions of the CS protein, were synthesized using solid phase Fmoc chemistry. The crude polypeptides were purified by a combination of size exclusion chromatography and RP-HPLC. Sera of mice immunized with the free polypeptides emulsified in incomplete Freund's adjuvant strongly reacted with the synthetic polypeptides as well as with native CS protein as judged by ELISA and IFAT assays. Most importantly, these antisera inhibited the sporozoite invasion of hepatoma cells. In addition, sera derived from donors living in a malaria endemic area recognized the CS 104- and 102-mers. Conformational studies of the CS polypeptides were also performed by circular dichroism spectroscopy showing the presence of a weakly ordered structure that can be increased by addition of trifluoroethanol. The obtained results indicate that the synthetic CS polypeptides and the natural CS protein share some common antigenic determinants and probably have similar conformation. The approach used in this study might be useful for the development of a synthetic malaria vaccine.

Tyrosine protein kinases (TPKs) of the src family contain two major phosphoacceptor sites which are homologous to the Tyr 416 and Tyr 527 of pp60c-src. The former represents the main autophosphorylation sites of these enzymes, and its phosphorylation correlates with increased kinase activity. It has previously been demonstrated that the Src-like tyrosine kinase expressed by the oncogene lyn displays a high affinity toward the heptapeptide H-Glu-Asp-Asn-Glu-Tyr-Thr-Ala-OH, which reproduces the main autophosphorylation site of the Src family enzymes [Donella-Deana, A., Marin, O., Brunati, A.M. & Pinna, L.A. (1992) Eur. J. Biochem. 204, 1159-1163]. Our study was addressed to the synthesis of some derivatives of this sequence in order to obtain both peptide substrates suitable for the detection of the Src-like tyrosine kinase activity and active site-directed inhibitors specific for this class of enzymes. For this purpose we synthesized by classical solution methods the heptapeptide and its dimeric form. Moreover, in order to improve the proteolytic resistance of these peptides we also synthesized their cyclic derivatives and their N-terminal acetylated and C-terminal amidated analogs. The correlation between the different structural properties induced by the modifications of the native sequence and the propensity of the peptides to act as Lyn substrates was examined. The kinetic data obtained indicate that the extent of the peptide phosphorylation varies considerably depending on the flexibility and length of the analogs. While the cyclization and the C-terminal amidation of the heptapeptide are detrimental for the Lyn activity, dimeric derivatives display very favourable kinetic constants. In particular the cyclic dimer is an especially suitable substrate for the tyrosine kinase and a powerful inhibitor of both the phosphorylation activity of Lyn and the enzyme autophosphorylation.

The synthesis of peptides containing blocks of arginyl residues is proposed through amidination of the corresponding ornithyl analogs. In order to test this strategy the ornithyl analog of the C-terminal sequence 52--65 of galline was synthesized by the conventional method. The amidination reaction, performed on fragments of different length and ornithyl-residue content, quantitatively converts ornithines into arginines. The strategy proposed may represent a powerful tool for the synthesis of protamines and other basic proteins.

Far-ultraviolet difference absorption and circular dichroism (CD) spectra were recorded upon recombination of synthetic S-peptide analogs, i.e. 1epsilon, 7epsilon-diguanidino-[Tyr8]-, 1epsilon, 7epsilon-diguanidino-[Asn14]-, [Phe(F)8, Orn10]-, [Cha8, Orn10]- and 1epsilon, 7epsilon-diguanidono-S-peptide, with S-protein. The aromatic chromophores contributions to the absorption spectra in the 220-250 nm wavelengths interval strongly exceed the hyperchromism due to the random coil to right handled alpha-helix transition of the S-peptide, accompanying the association process. Contributions resulting from peptide transitions constitute a large portion of the total dichroism, nevertheless substitution of the non aromatic cycloexylalanine residue in position 8 of the S-peptide with phenylalanine, p-fluorophenylalanine and tyrosine leads to CD negative maxima located at 215, 215 and 212,5 nm, respectively. The strongest ellipticity increment (28%), relative to that of the Cha-derivative, was observed at 212,5 nm for the [Tyr8]-S-peptide analog, while in the narrow 222 nm range minimal differences were found upon insertion into the position 8 of the S-peptide of the above aromatic residues. Information derived from above data and from a comparison of RNAase A, S and S-protein CD spectra enabled us to assign the positive CD band at 240 nm in RNAase A spectrum to transitions of phenylalanines and inaccessible tyrosines.

Near-ultraviolet difference absorption and circular dichroism (CD) spectra were recorded upon recombination of synthetic S-peptide analogs, i.e. 1epsilon, 7epsilon-diguanidino-[Tyr8]-,1epsilon,7epsilon-diguanidono-[Asn14]-, [Phe(F)8, Orn10]- and 1epsilon, 7epsilon-diguanidino-S-peptide, with S-protein. Environmental alterations of Phe-8 in the S-peptide and Tyr-25 in the S-protein, derived from the association process, lead to strong optical signals whose location and magnitude were clearly defined by means of a comparative analysis of the above spectra. Additionally, the spectroscopic effects resulting from insertion of a tyrosyl residue into an hydrophobic environment in the presence or absence of hydrogen-bonding partners were identified and compared with similar findings obtained from the model compound p-cresol.