Thermodynamics of A beta(1-40) amyloid fibril elongation.
We describe herein the characterization of the equilibrium point for A beta(1-40) amyloid fibril elongation in phosphate-buffered saline at 37 degrees C. Seeded fibril elongation progresses rapidly to a reproducible end point of 0.7-1.0 microM unpolymerized monomeric peptide. This remaining monomeric material is functional, since after concentration it supports fibril elongation. Incubation of fibrils in the same buffer results in dissociation to a final monomer concentration in the same range. This robust C(r) value is equivalent to the A beta(1-40) fibril dissociation constant, Kd. The fact that a similar value for Kd is obtained from a ratio of dissociation and elongation rate constants further supports the view that these values are associated with a position of dynamic equilibrium and therefore are related to free energies of amyloid fibril elongation. The C(r) value reported here for wild-type A beta(1-40) fibrils corresponds to a free energy of fibril elongation of about -9 kcal/mol, a value similar to free energies of folding for small globular proteins. Elongation and dissociation of amyloid fibrils from point mutants of A beta(1-40) also yield C(r) values, different for different mutants, that reflect stabilizing/destabilizing effects. Interestingly, assembly of A beta(1-40) fibrils in the presence of a saturating concentration of the amyloid dye thioflavin T does not measurably affect fibril stability, in contrast to the commonly observed stabilization of globular proteins by ligand binding. The ability to quantify and compare amyloid fibril thermodynamic stabilities makes it possible to include fibrils, and potentially other aggregates, in quantitative descriptions of protein folding landscapes.
O'Nuallain
Brian
B
Graduate School of Medicine, University of Tennessee, 1924 Alcoa Highway, Knoxville, Tennessee 37920, USA.
Shivaprasad
Shankaramma
S
Kheterpal
Indu
I
Wetzel
Ronald
R
D016229:Amyloid beta-Peptides / Q000737:chemistry* / Q000235:genetics; D052160:Benzothiazoles; D002851:Chromatography, High Pressure Liquid; D041961:Deuterium Exchange Measurement; D007700:Kinetics; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics; D017354:Point Mutation; D017510:Protein Folding; D013816:Thermodynamics; D013844:Thiazoles / Q000737:chemistry
D016229:Amyloid beta-Peptides; D052160:Benzothiazoles; D010446:Peptide Fragments; D013844:Thiazoles; C078592:amyloid beta-protein (1-40); C009462:thioflavin T