Nucleation-dependent Aggregation Kinetics of Yeast Sup35 Fragment GNNQQNY

JOURNAL OF MOLECULAR BIOLOGY

卷:433

Article

Burra, Gunasekhar^1,2  Maina, Mahmoud B.^2,3  Serpell, Louise C.²  Thakur, Ashwani K.¹ 

[1] Indian Inst Technol Kanpur, Dept Biol Sci & Bioengn, Kanpur, Uttar Pradesh, India

[2] Univ Sussex, Sussex Neurosci, Sch Life Sci, Brighton, E Sussex, England

[3] Yobe State Univ, Coll Med Sci, Damaturu, Nigeria

关键词: nucleation kinetics;    aggregation;    amyloid;    seeding;    temperature;   

DOI  :  10.1016/j.jmb.2020.166732

来源: Elsevier

【 摘 要 】

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 similar to 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 beta-sheet rich structures displaying a characteristic cross-beta 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. (C) 2020 Elsevier Ltd. All rights reserved.

【 授权许可】

Free