Chiral and Structural Polymorphism of Fibril Architectures of Homologous Lysozymes

Abstract

Amyloid fibrils are fascinating and complex structures with the multilayered chiral organization. Using the multimodal methodology, including VCD, ECD, cryo‐EM, and TEM, we characterized in detail different levels of organization (secondary structure/protofilament/mesoscopic structure) of amyloid fibrils prepared from proteins highly homologous in the structure (hen egg white and human lysozymes). Our results demonstrate that small changes in the native protein structure or preparation conditions translate into significant differences in the handedness and architecture of the formed fibrils at various levels of their complexity. In particular, fibrils of hen egg white and human lysozymes obtained in vitro at the same preparation conditions, possess different secondary structure, protofilament twist and ultrastructure. Yet, formed fibrils adopted a relatively similar mesoscopic structure, as observed in high‐resolution 3D cryo‐EM, scarcely used up to now for fibrils obtained in vitro in denaturing condition. Our results add to other puzzling experiments implicating the indeterministic nature of fibril formation.

Chirality induction and amplification in supramolecular systems exhibiting vibrational optical activity

“…1). [42][43][44][45][46][47][48] Due to the 'superchiral' long-range organization of the amyloid fibrils, they provide a very intense chiroptical readout. The enhanced VCD is observed not only for fibrils formed from proteins, but also for short peptides forming long, multistrand and twisted fibrillary architectures.…”

“…The most characteristic VCD signal of amyloid fibrils is the amide I band, derived from the excitonically coupled CQO stretching vibrations. Several studies 43,44,46,52,53 have demonstrated that the sign of this coupled might be positive or negative and that some factors, such as pH, 52 a solvent 53 or agitation, 43 can reverse it. Currently, it is assumed that the sign of the amide I VCD couplet is associated with the protofilament twist, 46 making VCD a rather unique method providing information about the protofilament handedness, hence constituting an important complement to other chiroptical and microscopic techniques in the characterization of amyloid fibrils (Fig.…”

Exceptionally enhanced Raman optical activity (ROA) of amyloid fibrils and their prefibrillar states

“…For all three proteins, significant enhancement of VCD was previously reported. 3,7 We decided to explore and directly compare the ROA with VCD, electron circular dichroism (ECD) and transmission electron microscopy (TEM) data to control and characterize the amyloid fibril formation. The experimental details are given in the ESI; † briefly, the fibrils were prepared under the previously reported conditions, i.e., at pH of 2.0 and at the elevated temperature of 60 1C.…”

“…The experimental details are given in the ESI; † briefly, the fibrils were prepared under the previously reported conditions, i.e., at pH of 2.0 and at the elevated temperature of 60 1C. 3,7 The lysozyme fibrils were obtained using agitation at 1400 rpm, resulting in the formation of thicker (HEWL, HL) and shorter (HEWL) fibrils compared with non-agitated samples, 7 presumably increasing the signal intensity and minimizing the anisotropy that could lead to artifacts in the ROA spectra. For both homologous lysozymes, ROA was measured at various time points from the onset of protein fibrilization.…”

“…The sign of the couplet is attributed to the protofilament twist and can be reversed by the application of some factors, including pH 4–6 and, as recently shown, agitation. 7…”