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 ¹H and 2D ¹H-¹³C 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.

Figure 1

(A) Size-exclusion chromatogram for GB1HS#124^F26A during fibrillation at time points (I) 0 h or the start point of incubation (II) 12 h, (III) 36 h, and (IV) 84 h. (B) Elution profile of lysozyme (14.6 kDa) and wt GB1(6.2 kDa) run as standards to confirm the size of dimeric and monomeric species of GB1 HS#124^F26A

Figure 2

DLS profile of GB1 HS#124^F26A depicting lower molecular weight soluble species present at time points 0, 12, 36, and 84 h of fibrillation.

Figure 3

1D ¹H NMR spectra of GB1HS#124^F26A at 0, 12, 36, and 84 h of fibrillation at 58 °C and pH = 5.5.

Figure 4

Changes in the mean diffusion coefficient during fibrillation measured at time point 0, 12, 36, and 84 h, respectively.