Vibrational spectroscopy characterization of self-assembling oligopeptides.

In the emerging field of tissue engineering, the development of synthetic materiate promoting cells growth has led to the study of functionalised biomimetic materiate and in particular to investigations on regular alternating polar/non-polar oligopeptides such as EAK-16, AEAEAKAKAEAEAKAK, first synthesised by Zhang et al. The characteristic of EAK-16 is to have a preferenti al (i-sheet structure, to be resistant to proteolytic cleavage and to self-assemble into an insoluble macroscopic membrane. Its ability to create stable self-assembling layers derives from hydrophobic interactions between the -Cth groups of non-ionic residues and complementary ionie bonds between acidic and basic amino acids: this stability can be enhanced by the regulation of pH and the presence of monovalent metallic ions. In order to evaluate the ability to form self-assembled layers on oxidised titanium surfaces, we investigated 7 different oligopeptides (16 to 19 residues) derived from EAK-16 but modifìed in their sequence by substitution of acid, basic and neutral amino acid or by the addition at the N-terminus of the RGD sequence, able to control osteoblast adhesion. The techniques used to determine the structure of the oligopeptides were IR and Raman vibrational spectroscopies, which can provide useful information on the secondary structure of the peptides, both on the qualitative and the quantitative aspect, by the help of different amide stretching modes. The peptides were examined as synthesised and after solubilisation in saline phosphate buffer (pH=7.4) and lyophilisation, as well as after deposition on oxidised titanium substrates. A quantitative evaluation of the secondary structure of the oligopeptides was obtained by fitting the Raman and IR amide I bands. As regards the just-synthesised peptides, those with acidic substitution, basic substitution or both resulted to have a prevalent beta-sheet structure, as revealed by the position of the amide I band at 1670-1673 cm-1 in their Raman spectra and at 1694-1697 cm-1 and 1620-1626 cm-1 in the IR ones. The substitution of the non polar amino acid induced in the fìrst case an increase of alfa-helix conformation while in the second one allowed a higher order of the hydrophobic component. Mainly alfa-helix or mixed content was found in the peptides containing the RGD head, as revealed by the presence of Raman and IR bands at 1659 and 1640 cm-1, respectively. Also the vNH stretching values confìrmed the results on the prevailing conformation of the peptides: peptide 8 showed the highest vNH value and the largest width of the corresponding band, indicating a less ordered structure. After treatment for 6 hours in saline phosphate buffer and lyophilisation, for all peptides a decrease in conformational order was observed, although the prevalent structure remained beta-sheet for all peptides without the RGD head. Of the two oligopeptides containing the RGD sequence, the one with the most regular sequence changed its secondary structure to a prevalent beta-sheet one while that having a "scrambled" structure became prevalently unordered with a lower beta-sheet content. Preliminary studies on EAK-16 and the scrambled peptide deposited on oxidised porous titanium plates showed that the TiO2 surface induced the same conformational changes as the solubilisation/lyophilisation treatment.