Nucleation-dependent Aggregation Kinetics of Yeast Sup35 Fragment GNNQQNY. Issue 3 (5th February 2021)

• Record Type: Journal Article
• Authors: Burra, Gunasekhar; Maina, Mahmoud B.; Serpell, Louise C.; Thakur, Ashwani K.
• Is Part Of: Journal of molecular biology. Volume 433:Issue 3(2021)
• Publication Date: 2021-02-05
• Subjects: nucleation kinetics -- aggregation -- amyloid -- seeding -- temperature; CR Congo-red -- Cryo-EM Cryo-electron microscopy -- ESI Electrospray ionization -- FPLC Fast protein liquid chromatography -- FTIR Fourier-transform infrared spectroscopy -- HCl Hydrogen chloride -- LS Light scattering -- PBS Phosphate-buffered saline -- RP-HPLC Reversed-phase high performance liquid chromatography -- SAXS Small angle X-ray scattering -- SEC Size-exclusion chromatography -- TEM Transmission electron microscopy -- ThT Thioflavin T -- ToF Time-of-flight; Molecular biology -- Periodicals; Biology -- Periodicals; Biochemistry -- Periodicals; Bacteriology -- Periodicals; Molecular Biology -- Periodicals; Biochemistry -- Periodicals; Biologie moléculaire -- Périodiques; Biologie -- Périodiques; Biochimie -- Périodiques; Moleculaire biologie; Biochemistry; Biology; Molecular biology; Periodicals; 572.805
• DOI: 10.1016/j.jmb.2020.166732
• Languages: English
• ISSNs: 0022-2836

Abstract

Graphical abstract: This study provides experimental evidences for the Nucleation-dependent aggregation kinetics followed by the well studied N-terminal 7GNNQQNY13 sequence of yeast Sup35 prion protein. Highlights: GNNQQNY follows nucleation-dependent aggregation kinetics. It forms a critical nucleus of size ~7 monomers during self-assembly. The size of the nucleus formed is proportional to the reaction temperature. Mature GNNQQNY fibers exhibit no significant oxidative stress or cytotoxicity. 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.