RESUMO
Recent therapeutic strategies suggest that small peptides can act as aggregation inhibitors of monomeric amyloid-ß (Αß) by inducing structural rearrangements upon complexation. However, characterizing the binding events in such dynamic and transient noncovalent complexes, especially in the presence of natively occurring metal ions, remains a challenge. Here, we deploy a combined transition metal ion Förster resonance energy transfer (tmFRET) and native ion mobility-mass spectrometry (IM-MS) approach to characterize the structure of mass- and charge-selected Aß complexes with Cu(II) ions (a quencher) and a potential aggregation inhibitor, a small neuropeptide named leucine enkephalin (LE). We show conformational changes of monomeric Αß species upon Cu(II)-binding, indicating an uncoiled N-terminus and a close interaction between the C-terminus and the central hydrophobic region. Furthermore, we introduce LE labeled at the N-terminus with a metal-chelating agent, nitrilotriacetic acid (NTA). This allows us to employ tmFRET to probe the binding even in low-abundance and transient Aß-inhibitor-metal ion complexes. Complementary intramolecular distance and global shape information from tmFRET and native IM-MS, respectively, confirmed Cu(II) displacement toward the N-terminus of Αß, which discloses the binding region and the inhibitor's orientation.