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Please use this identifier to cite or link to this item: http://hdl.handle.net/10481/31076

Title: Characterization of Oligomers of Heterogeneous Size as Precursors of Amyloid Fibril Nucleation of an SH3 Domain: An Experimental Kinetics Study
Authors: Ruzafa, David
Morel, Bertrand
Varela Álvarez, Lorena
Azuaga Fortes, Ana Isabel
Conejero-Lara, Francisco
Issue Date: 2012
Abstract: Understanding the earliest molecular events during nucleation of the amyloid aggregation cascade is of fundamental significance to prevent amyloid related disorders. We report here an experimental kinetic analysis of the amyloid aggregation of the N47A mutant of the α-spectrin SH3 domain (N47A Spc-SH3) under mild acid conditions, where it is governed by rapid formation of amyloid nuclei. The initial rates of formation of amyloid structures, monitored by thioflavine T fluorescence at different protein concentrations, agree quantitatively with high-order kinetics, suggesting an oligomerization pre-equilibrium preceding the rate-limiting step of amyloid nucleation. The curves of native state depletion also follow high-order irreversible kinetics. The analysis is consistent with the existence of low-populated and heterogeneous oligomeric precursors of fibrillation that form by association of partially unfolded protein monomers. An increase in NaCl concentration accelerates fibrillation but reduces the apparent order of the nucleation kinetics; and a double mutant (K43A, N47A) Spc-SH3 domain, largely unfolded under native conditions and prone to oligomerize, fibrillates with apparent first order kinetics. On the light of these observations, we propose a simple kinetic model for the nucleation event, in which the monomer conformational unfolding and the oligomerization of an amyloidogenic intermediate are rapidly pre-equilibrated. A conformational change of the polypeptide chains within any of the oligomers, irrespective of their size, is the rate-limiting step leading to the amyloid nuclei. This model is able to explain quantitatively the initial rates of aggregation and the observed variations in the apparent order of the kinetics and, more importantly, provides crucial thermodynamic magnitudes of the processes preceding the nucleation. This kinetic approach is simple to use and may be of general applicability to characterize the amyloidogenic intermediates and oligomeric precursors of other disease-related proteins.
Sponsorship: This work was financed by the Andalucía Government (grant FQM-02838), the Spanish Ministry of Science and Innovation (grant BIO2009-07317), and the European Regional Development Fund of the European Union. D. Ruzafa is recipient of a research fellowship from the F.P.U. program of the Spanish Ministry of Education. L. Varela is financed by the G.R.E.I.B. program of the University of Granada.
Publisher: Public Library of Science (PLOS)
Description: Correction: Characterization of Oligomers of Heterogeneous Size as Precursors of Amyloid Fibril Nucleation of an SH3 Domain: An Experimental Kinetics Study. PLoS ONE 9(1): 10.1371/annotation/dbb84118-9ada-43e4-8734-8f8322be1a59. doi: 10.1371/annotation/dbb84118-9ada-43e4-8734-8f8322be1a59
Keywords: Elution
Glycine
Nucleation
Oligomers
Polymerization
Protein aggregation
Protein structure
Thermodynamics
URI: http://hdl.handle.net/10481/31076
ISSN: 1932-6203
Rights : Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License
Citation: Ruzafa, D.; et al. Characterization of Oligomers of Heterogeneous Size as Precursors of Amyloid Fibril Nucleation of an SH3 Domain: An Experimental Kinetics Study. Plos One, 7(11): e49690 (2012). [http://hdl.handle.net/10481/31076]
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