Changes of Secondary Structure Detected by Laser Raman Spectroscopy in Model Peptides of Human αA-Crystallin Due to Substitution of D-Aspartyl Residues for L-Aspartyl Residues

Abstract

Purpose: We have found that two aspartyl (Asp-151 and Asp-58) residues of αA-crystallin are inverted and isomerized to the biologically uncommon D-β-Asp residues during aging. In order to elucidate the correlation between the formation of the D-β-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 αA-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-α-, D-α, L-β and D-β-aspartyl residues. These peptides were subjected to Raman measurement. Results: The Raman spectrum of the L-α-Asp T18 peptide measured as dry powder revealed that the secondary structure of this peptide is mainly anti-parallel β-sheet. The main structure of the D-β-Asp T18 peptide when in dry powder form was altered to an α-helix and/or random structure. The main structure of L-α-Asp T18 peptide when measured in aqueous solution also converted to an α-helix and/or random structure. The conversion of L-α-to D-β-Asp in T6 peptides when in dry powder form revealed no alteration of secondary β-sheeted structure. Conclusion: Raman spectroscopy clearly revealed a large conformational change in the secondary structure of T18 peptide caused by substitution of normal L-α-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.