Nucleation-dependent aggregation kinetics of yeast Sup35 fragment GNNQQNY

Burra, Gunasekhar, Maina, Mahmoud B, Serpell, Louise C and Thakur, Ashwani K (2021) Nucleation-dependent aggregation kinetics of yeast Sup35 fragment GNNQQNY. Journal of Molecular Biology, 433 (3). p. 166732. ISSN 0022-2836

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Abstract

An N-terminal hepta-peptide sequence of yeast prion protein Sup35 with the sequence GNNQQNY is widely used as a model system for amyloid fibril formation. In this study, we used a reproducible solubilisation protocol that allows the generation of a homogenous monomeric solution of GNNQQNY to uncover the molecular details of its self-assembly mechanism. The aggregation kinetics data show that the GNNQQNY sequence follows nucleation-dependent aggregation kinetics with a critical nucleus of size ~7 monomers and that the efficiency of nucleation were found to be inversely related to the reaction temperature. The 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 fluorescence, 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 did not exert significant oxidative stress or cytotoxicity upon incubating with differentiated SHSY5Y cells. To our knowledge, this is the first study to experimentally validate previous nucleus size predictions based on theoretical and molecular dynamics simulations. These findings provide the basis for understanding the kinetics and thermodynamics of amyloid nucleation and elongation of amyloidogenic proteins/peptides associated with many systemic and neurodegenerative diseases.

Item Type: Article
Keywords: aggregation, amyloid, nucleation kinetics, seeding, temperature, Amino Acid Sequence, Amyloid, Amyloidogenic Proteins, Cell Survival, Fluorescent Antibody Technique, Fungal Proteins, Kinetics, Peptide Fragments, Protein Aggregates, Protein Transport, Solubility, Spectrum Analysis, Thermodynamics, Yeasts
Schools and Departments: School of Life Sciences > Biochemistry
School of Life Sciences > Neuroscience
SWORD Depositor: Mx Elements Account
Depositing User: Mx Elements Account
Date Deposited: 16 Sep 2021 13:51
Last Modified: 04 Dec 2021 02:00
URI: http://sro.sussex.ac.uk/id/eprint/101711

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