An N-terminal hepta-peptide sequence of yeast prion protein Sup35 with the sequence GNNQQNY serves as an ideal model for structural understanding of amyloid assembly and kinetics. In this study, we used a reproducible solubilisation protocol that allows the generation of homogenous monomeric solution of GNNQQNY to understand the molecular details of its self-assembly mechanism. The aggregation kinetics data show that the GNNQQNY sequences follow nucleation-dependent aggregation kinetics with a critical nucleus of size ~7 monomers and that the size and efficiency of nucleation was found to be inversely related to the reaction temperature. The generated nucleus reduces the thermodynamic energy barrier by acting as a template for further self-assembly and results in highly ordered amyloid fibrils. The fibers grown at different temperatures showed similar Thioflavin T positivity, Congo red binding and β-sheet rich structures displaying a characteristic cross-β diffraction pattern. These aggregates also share morphological and structural identity with those reported earlier. The mature GNNQQNY fibers exerted no significant oxidative stress or cytotoxicity upon incubating with differentiated SHSY5Y cells. To our knowledge, this is the first study to experimentally validate previous predictions based on theoretical and molecular dynamics simulations. These findings will provide the basis for understanding the kinetics and thermodynamics of amyloid nucleation and elongation of amyloidogenic systems associated with many systemic and neurodegenerative diseases. ### Competing Interest Statement The authors have declared no competing interest.
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