A Double Mutant Cycle Involving the Charged Residues of Amyloid Beta

Abstract

A critical difference between the fibril structures of Amyloid beta 40 (Aβ40) and its more toxic variant Abeta42 (Aβ42), associated with Alzheimer's disease, lies in the nature of a specific salt bridge involving Lysine-28 (K28). In Aβ42, it has a salt bridge with the C-terminal negative charge, while in Aβ40 it has the bridge with Aspartate-23 (D23). Interestingly, Aβ40 oligomers, which are aggregation intermediates known to be more toxic than the fibrils, do not have the D23-K28 salt bridge. So we ask whether transient interactions between K28 and the C-terminal of Aβ40, make the oligomers similar to the more toxic Aβ42. We use the powerful technique of ‘double mutant cycle’ to probe if K28 and the terminal COO- interact directly or indirectly during any stage of the aggregation process, and how that may influence the aggregation and toxicity of the peptide. We acetylate K28 and amidate the C-terminal, separately and also in the same mutant, and probe the aggregation kinetics, final solubility, secondary structure, membrane affinity and toxicity of the 4 variants. We find that the K28 acetylation has a very strong effect on the solubility, while amidation of the C-terminal has a mild but opposite effect. While K28 acetylation hardly affects the membrane affinity, amidation increases it sharply. However, the secondary structure of the amidated Abeta40 differs from the wild-type in the fibrillary state. Interestingly, both C-terminal amidation and K28-acetylation decrease the toxicity of the peptide considerably. Our results show that the K28 and the terminal COO- have independent but strong effects on the properties of the peptide. Moreover, the results prove the futility of focusing on reagents that just reduce aggregation propensity, since the toxicity of the peptide can vary independently.