Changes of secondary structure detected by laser Raman spectroscopy in model peptides of human alphaA-crystallin due to substitution of D-aspartyl residues for L-aspartyl residues.

PURPOSE We have found that two aspartyl (Asp-151 and Asp-58) residues of alphaA-crystallin are inverted and isomerized to the biologically uncommon D-beta-Asp residues during aging. In order to elucidate the correlation between the formation of the D-beta-Asp isomer and the environment surrounding the Asp in the protein, we performed a Raman spectroscopic study using two synthetic peptides: T6 peptide containing Asp-58, and T18 peptide, containing Asp-151, which correspond to the tryptic peptides of human alphaA-crystallin. METHODS Both T6 (Thr-Val-Leu-Asp(58)-Ser-Gly-Ile-Ser-Glu-Val-Arg) and T18 (Ile-Gln-Thr-Gly-Leu-Asp(151)-ala-thr-his-ala-Glu-Arg) peptides were synthesized with four optical isomers which have L-alpha-, D-alpha, L-beta and D-beta-aspartyl residues. These peptides were subjected to Raman measurement. RESULTS The Raman spectrum of the L-alpha-Asp T18 peptide measured as dry powder revealed that the secondary structure of this peptide is mainly anti-parallel beta-sheet. The main structure of the D-beta-Asp T18 peptide when in dry powder form was altered to an alpha-helix and/or random structure. The main structure of L-alpha-Asp T18 peptide when measured in aqueous solution also converted to an alpha-helix and/or random structure. The conversion of L-alpha-to D-beta-Asp in T6 peptides when in dry powder form revealed no alteration of secondary beta-sheeted structure. CONCLUSION Raman spectroscopy clearly revealed a large conformational change in the secondary structure of T18 peptide caused by substitution of normal L-alpha-Asp to biologically uncommon Asp-isomers. This result indicates that the inversion of an amino acid in a protein greatly affects the secondary structure of the protein.