The misfolding and aggregation of various peptides and proteins into amyloid fibrils are key molecular processes in a variety of neurodegenerative pathologies, including Alzheimer’s and Parkinson’s diseases. Throughout the fibril formation process, intermediate misfolded protein oligomers are continuously formed. These small, transient aggregates are highly cytotoxic and thought to play an important role in the onset and development of numerous protein misfolding diseases. Previous research suggests that metal ions contribute to conformational changes in the pathway to forming amyloid fibrils. To investigate the biophysical properties of oligomers formed in the presence of cationic ligands, we leveraged a model amyloid system from the 91-residue N-terminal domain of the [NiFe]-hydrogenase maturation factor HypF (HypF-N), found in E. Coli, as this system forms homogenous populations of oligomeric species under controlled solution conditions. By introducing K⁺, Na⁺, Li⁺, Ca²⁺, Mg²⁺, and Zn²⁺ into solution at the beginning of the controlled oligomerization for HypF-N, we observed the formation of HypF-N oligomers with differential structural, chemical, physical, and toxicological properties. Specifically, we explored ion-induced differences in oligomer β-sheet content, solvent exposed hydrophobicity, size, and yields. Relating these biophysical readouts to the ability of the various oligomers to induce cellular dysfunction, as measured by the MTT assay and the Ca²⁺ influx assay, revealed that size is inversely correlated with cytotoxicity, while hydrophobicity is positively correlated with cytotoxicity. These findings elucidate the properties that promote protein misfolded oligomers to become cytotoxic to cells and suggest conformations of aggregates that may be stabilized by endogenous levels of specific metal cations naturally present in the brain during pathology.

Ryan Limbocker

United States Military Academy at West Point

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