Detergent-like Toxicity Mechanism of Amyloid Cylindrins Revealed by Cross-Link Spin Labeling Electron Spin Resonance

Amyloid oligomers are considered to be the most toxic species in neurodegenerative diseases, such as Alzheimer's, and Parkinson's, among others. A major challenge in studying amyloid oligomers has been their heterogeneous character, coupled with a lack of methods that can probe targeted, localized structural information. In this paper, we show that in detergent micelles, the oligomeric state of amyloid cylindrin peptide K11 V increases from a hexamer to an octamer. This reveals a detergent-like toxicity mechanism of the K11 V peptide, in which the octamer-form extracts lipids from biological membranes. The findings are confirmed by Alphafold simulations, showing that the octamer has a β-barrel structure, with a pore stabilized by using the unbranched hydrophobic tails of the detergent molecules. The experimental results are obtained by a unique electron spin resonance (ESR)-based methodology that can be applied to amyloidogenic proteins to uncover inaccessible amyloid oligomer structures. The process involves (1) stabilization of the minimal amyloid oligomer domain, using a cross-linking bifunctional nitroxide spin label, combined with (2) an oligomer stabilizing mutation, (3) DEER spectroscopy, which can be performed in a native environment, and (4) T1-edited DEER measurements, which can validate structure in samples with heterogeneous populations, combined with (5) a model-free distance reconstruction approach that can analyze distance distributions with inverted distance components, ideal for analysis of T1-edited DEER.