Application of the Double-Mutant Cycle Strategy to Protein Aggregation Reveals Transient Interactions in Amyloid-β...

Transient oligomeric intermediates in the peptide or protein aggregation pathway are suspected to be the key toxic species in many amyloid diseases, but deciphering their molecular nature has remained a challenge. Here we show that the strategy of "double-mutant cycles", used effectively in probing protein-folding intermediates, can reveal transient interactions during protein aggregation. It does so by comparing the changes in thermodynamic parameters between the wild type, and single and double mutants. We demonstrate the strategy by probing the possible transient salt bridge partner of lysine 28 (K28) in the oligomeric states of amyloid β-40 (Aβ₄₀), the putative toxic species in Alzheimer's disease. In mature fibrils, the binding partner is aspartate 23. This interaction differentiates Aβ₄₀ from the more toxic Aβ₄₂, where K28's binding partner is the C-terminal carboxylate. We selectively acetylated K28 and amidated the C-terminus of Aβ₄₀, creating four distinct variants. Spectroscopic measurements of the kinetics and thermodynamics of aggregation show that K28 and the C-terminus interact transiently in the early phases of the Aβ₄₀ aggregation pathway. Hydrogen-deuterium exchange mass spectrometry (using a simple analysis method that we introduce here that takes into account the isotopic mass distribution) supports this interpretation. It is also supported by cellular toxicity measurements, suggesting possible similarities in the mechanisms of toxicity of Aβ₄₀ oligomers (which are more toxic than Aβ₄₀ fibrils) and Aβ₄₂. Our results show that double-mutant cycles can be a powerful tool for probing transient interactions during protein aggregation.