The C-terminal domain of SARS-CoV main protease (Mpro-C) can form 3D domain-swapped dimer by exchanging the α1-helices fully buried inside the protein hydrophobic core, under non-denaturing conditions. 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. 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 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.
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. 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. Non-physiological destabilizing conditions used in many in vitro aggregation studies are shown to yield qualitatively different, highly misfolded amyloid-like fibrils. Surprisingly, solid-state NMR reveals that these amorphous deposits have a high degree of structural homogeneity at the atomic level and that the aggregated protein retains a native-like conformation, with no evidence for large-scale misfolding.
At physiological pH, the protein forms aggregates that look amorphous and disordered by electron microscopy, reminiscent of the reported formation of amorphous deposits by other crystallin mutants. We describe the structure and morphology of aggregates formed by the P23T human γD-crystallin mutant associated with congenital cataracts. The development of new treatments is hindered by uncertainty about the nature of the aggregates and their mechanism of formation. We discuss these observations in light of recent work on related amyloid-forming proteins that have been argued to follow similar mechanisms and how this may have implications for the role of domain-swapping propensities for amyloid formation.Ĭataracts cause vision loss through the large-scale aggregation of eye lens proteins as a result of ageing or congenital mutations. This observation contrasts with predictions that native structure, and in particular intermolecular β-strand interactions seen in the dimeric state, may be preserved in "domain-swapping" fibrils. Our results reveal a highly rigid fibril structure that lacks mobile domains and indicate a parallel in-register β-sheet structure and a general loss of native conformation within the mature fibrils. We employ magic angle spinning solid-state NMR and other methods to examine key structural features of these fibrils. Under native conditions this mutant adopts a domain-swapped dimer, and it also forms amyloid-like fibrils, seemingly in correlation to its domain-swapping ability. One example is an amyloid-forming mutant of the immunoglobulin binding domain B1 of streptococcal protein G, which in its native conformation consists of a four-stranded β-sheet and one α-helix. Intramolecular β-sheet contacts present in the monomeric state could constitute intermolecular β-sheets in the dimeric and fibrillar states.
Certain mechanisms have been proposed based on the three-dimensional or runaway domain swapping, inspired by the fact that some proteins show an apparent correlation between the ability to form domain-swapped dimers and a tendency to form fibrillar aggregates. The formation of amyloid-like fibrils is characteristic of various diseases, but the underlying mechanism and the factors that determine whether, when, and how proteins form amyloid, remain uncertain.
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