Modulation of the stability of amyloidogenic precursors by anion binding strongly influences the rate of amyloid nucleation.
A deep understanding of the physicochemical factors modulating amyloid aggregation of proteins is crucial to develop therapeutic and preventive approaches for amyloid-related diseases. The earliest molecular events of the aggregation cascade represent some of the main targets as indicated by the toxic nature of certain early oligomers. Here, we study how different types of salt ions influence the kinetics of amyloid assembly of the N47A mutant α-spectrin SH3 domain using a battery of techniques. The salts influenced aggregation rates to different extents without altering the overall mechanism and the high apparent order of the experimental kinetics. A quantitative analysis of the initial aggregation rates measured by thioflavine-T fluorescence using a simple nucleation model allowed us to estimate the kinetic and thermodynamic magnitudes of crucial aggregation precursors, as well as to evaluate the impact of each type of ion on the earliest amyloid nucleation stages. Whilst cations did not have any noticeable effect under our experimental conditions, anions stabilized an amyloidogenic intermediate state and also increased the rate of the conformational conversion from dynamic oligomers to amyloid nuclei, resulting in a strong acceleration of the nucleation process. Anions appear to act by preferential binding to the amyloidogenic intermediate state, thus enhancing its population and subsequent oligomerization. Overall, our results contribute to the rationalization of the effect of ions on the amyloid nucleation stage and give insight into the properties of the crucial intermediate precursors of amyloid aggregation.
Departamento de Química Física e Instituto de Biotecnología, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain. bmorel@ugr.es.
David
D
Ruzafa
Francisco
F
Conejero-Lara
Bertrand
B
Morel
D000682:Amyloid / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000838:Anions / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation; D055503:Protein Multimerization; D055550:Protein Stability; D012492:Salts / Q000378:metabolism*; D013049:Spectrin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013816:Thermodynamics; D018909:src Homology Domains
D000682:Amyloid; D000838:Anions; D012492:Salts; D013049:Spectrin