Biophysical Investigation of the Interplay between the Conformational Species of Domain-Swapped GB1 Amyloid Mutant through Real–Time Monitoring of Amyloid Fibrillation

Abstract

Mutant polypeptide GB1HS#124 F26A , which is known to aggregate into amyloid-like fibrils, has been utilized as a model in this study for gaining insights into the mechanism of domain-swapped aggregation through real-time monitoring. Size exclusion with UV monitoring at 280 nm and dynamic light scattering (DLS) profiles through different time points of fibrillation reveal that the dimer transitions into monomeric intermediates during the aggregation, which could further facilitate domain swapping to form amyloid fibrils. The 1D 1 H and 2D 1 H– 13 C HSQC nuclear magnetic resonance (NMR) spectra profiling through different time points of fibrillation reveal that there may be some other species present along with the dimer during aggregation which contribute to different trends for the intensity of protons in the spectral peaks. Diffusion NMR reveals changes in the mobility of the dimeric species during the process of aggregation, indicating that the dimer gives rise to other lower molecular weight species midway during aggregation, which further add up to form the oligomers and amyloid fibrils successively. The present work is a preliminary study which explores the possibility of utilizing biophysical methods to gain atomistic level insights into the different stages of aggregation.

New Insights into the Structural and Binding Properties on Aβ Mature Fibrils Due to Histidine Protonation Behaviors

“…This pathway does not have intact Aβ fragments generated. (2) Pathogenic pathway: APP is sheared by β-secretases and γ-secretases, ,, and then, intact Aβ­(1–40/42) fragments are generated. , As an important indicator peptide, the Aβ fragment composition is about 90% of Aβ(1–40) and 10% of Aβ(1–42). − Importantly, the free lipids have been shown to play important roles in the lipid-membrane complex (lipid chaperone hypothesis) since Aβ is cleaved in the membrane, suggesting that free lipid molecules, not bound to membranes, affect the aggregation of amyloidogenic peptides. − It is noticeable that the aggregation mechanism of Aβ fragments is crucial for understanding the pathogenesis of AD. , Surely, there is a fragile dynamic balance between Aβ production and Aβ clearance. ,, When the crucial balance is out of whack, Aβ peptides will aggregate together from monomers to oligomers, , where the related toxicity is considered to be key contributor toward amyloid disease, , and eventually mature into fibril. Those oligomers and mature fibrils can impair neuronal signaling as well as cause neuronal cell death. , …”

“…19,20 Surely, there is a fragile dynamic balance between Aβ production and Aβ clearance. 10,21,22 When the crucial balance is out of whack, Aβ peptides will aggregate together from monomers to oligomers, 23,24 where the related toxicity is considered to be key contributor toward amyloid disease, 25,26 and eventually mature into fibril. Those oligomers and mature fibrils can impair neuronal signaling as well as cause neuronal cell death.…”