Site-Specific Structural Changes in Unmodified and Pyroglutamylated Amyloid Beta Peptide by Isotope-Edited Ftir

Abstract

Amyloid beta-peptide (Abeta) forms cytotoxic assemblies that contribute to Alzheimer's disease. Recent evidence indicates that prefibrillar aggregates and not the fibrillar deposits exert the main toxic effect. In addition, naturally occurring N-terminally truncated and pyroglutamylated peptide (pE-Abeta) displays augmented cytotoxicity by an unknown mechanism. This study examines the conformational changes in both unmodified Abeta and pE-Abeta upon exposure to an aqueous environment. FTIR and circular dichroism were used to identify alpha-helix-to-beta-sheet conformational transitions of both peptides during aggregation. To gain site-specific structural information, the peptides were 13C,15N-labeled at residues 16-18 (KLV) or 36-39 (VGGV), followed by FTIR analysis. The peptides dried from hexafluoroisopropanol were alpha-helical (amide I peak at 1660-1657 cm-1) and showed negligible intensity of the labeled segments around 1615 cm-1, suggesting that in alpha-helical conformation the labeled amide groups behave like isolated oscillators. Upon addition of aqueous buffer (pH 7.2) both peptides rapidly adopted beta-sheet structure (amide I peak at 1637-1629 cm-1), with disproportionally prominent components around 1604-1597 cm-1 generated by the labeled segments. The intensity and the frequency of the amide I mode of the isotope-labeled segments suggest 12C-13C vibrational coupling, consistent with formation of antiparallel beta-sheet structures. Moreover, the amide I contours of the peptides under near-physiological and low ionic strength conditions were significantly different; both peptides exhibited an increased alpha-helical and decreased beta-sheet propensity under low ionic strength conditions, indicating a strong influence of the ionic strength on the aggregation kinetics and accompanying structural changes. Ongoing studies focus on structural differences between the unmodified Abeta and pE-Abeta peptides as well as their mutual structural effects when combined at various molar ratios, in an attempt to understand the structural basis of the elevated cytotoxicity of pE-Abeta.