Characterizing the Assembly of the Sup35 Yeast Prion Fragment, GNNQQNY: Structural Changes Accompany a Fiber-to-Crystal Switch

Abstract

Amyloid-like fibrils can be formed by many different proteins and peptides. The structural characteristics of these fibers are very similar to those of amyloid fibrils that are deposited in a number of protein misfolding diseases, including Alzheimer's disease and the transmissible spongiform encephalopathies. The elucidation of two crystal structures from an amyloid-like fibril-forming fragment of the yeast prion, Sup35, with sequence GNNQQNY, has contributed to knowledge regarding side-chain packing of amyloid-forming peptides. Both structures share a cross-beta steric zipper arrangement but vary in the packing of the peptide, particularly in terms of the tyrosine residue. We investigated the fibrillar and crystalline structure and assembly of the GNNQQNY peptide using x-ray fiber diffraction, electron microscopy, intrinsic and quenched tyrosine fluorescence, and linear dichroism. Electron micrographs reveal that at concentrations between 0.5 and 10 mg/mL, fibers form initially, followed by crystals. Fluorescence studies suggest that the environment of the tyrosine residue changes as crystals form. This is corroborated by linear dichroism experiments that indicate a change in the orientation of the tyrosine residue over time, which suggests that a structural rearrangement occurs as the crystals form. Experimental x-ray diffraction patterns from fibers and crystals also suggest that these species are structurally distinct. A comparison of experimental and calculated diffraction patterns contributes to an understanding of the different arrangements accessed by the peptide.

Factors That Drive Peptide Assembly and Fibril Formation: Experimental and Theoretical Analysis of Sup35 NNQQNY Mutants

“…Previous works on CGNNQQNY in water (same solvent condition as ours) yielded aggregates in less than 1 week at a similar concentration (500−600 μM), 41 and GNNQQNY aggregated at time t = 0 at higher concentrations (≈ 3.5 mM). 40 The discrepancy in aggregation propensity of the short and long versions of the Sup35 fragment suggests that the glycine residue plays a role or the overall peptide must reach a certain length (number of residues) to become aggregationprone, as in the cases of polyglutamine and polyalanine. 13 In other words, a loss of glycine residue may shift the nucleation stage to a larger size oligomer.…”

“…However, the presence of abundant β-sheet conformations or pre-existing steric zipper structures observed by these methods at small oligomer sizes does not directly imply a high aggregation propensity because the internal rearrangement at large oligomer sizes may be very slow or kinetically inaccessible. 63 Some fibril-crystal distinctions in the packing of the peptide chains within the fiber and crystal forms of GNNQQNY have been shown by studies using diffraction analysis 40 and magic-angle-spinning NMR. 66 A recent elegant model proposed by Knowles and coworkers 67 has addressed the untwisting phenomenon of helical steric zipper during the transition from protofilaments to crystalline phase, which accounts for the discrepancies among the results obtained from measuring the protofilaments and the microcrystals.…”

“…The choice of solvent is consistent with previous work done by our group 9 and others. 40,41 Instrumentation. Ion-Mobility Mass Spectrometry (IM-MS).…”

Structural determinants in a library of low molecular weight gelators

“…Previous studies have shown that CD spectra collected from fibrillar samples may exhibit LD artefacts as a result of fibril alignment. 33,34 Cuvette rotation confirmed there was no significant directional signal dependence (LD) in any of the samples so the spectra are true CD spectra of the dipeptide self-assembled structures ( Figure 3). An isolated naphthalene chromophore exhibits absorbance signals from 200 -230 nm (long axis polarized with maximum at 225 nm) and 240 -290 nm (short axis polarized).…”

The diversity and utility of amyloid fibrils formed by short amyloidogenic peptides

“…The crystals have a hydrogen bonding direction, which is attributed to the fibre axis and the peptides are arranged within the crystallographic structure to form steric zippers in which the side chains associate and interdigitate [10,38]. Further studies using solid-state NMR [39], X-ray fibre diffraction [40] and small angle X-ray scattering [41] revealed that the fibrillar structure differs from the arrangement with the crystal packing. However, many short peptide structures have been solved crystallographically [10,42 -44], contributing valuable understanding of how sequence contributes to stability within the amyloid architecture.…”