Since polyglutamine (polyQ) aggregate formation has been implicated as playing an important role in expanded CAG repeat diseases, it is important to understand the biophysics underlying the initiation of aggregation. Previously we showed that relatively long polyQ peptides aggregate by nucleated growth polymerization and a monomeric critical nucleus. We show here that, over a short repeat length range from Q26 to Q23, the size of the critical nucleus for aggregation increases from monomeric to dimeric to tetrameric. This variation in nucleus size suggests a common duplex anti-parallel β-sheet framework for the nucleus, and further supports the feasibility of an organized monomeric aggregation nucleus for longer polyQ repeat peptides. The data also suggest that a change in aggregation nucleus size may play a role in the pathogenicity of polyQ expansion in this series of familial neurodegenerative diseases.

Polyglutamine expansion within the exon1 of huntingtin leads to protein misfolding, aggregation, and cytotoxicity in Huntington's disease. This incurable neurodegenerative disease is the most prevalent member of a family of CAG repeat expansion disorders. Although mature exon1 fibrils are viable candidates for the toxic species, their molecular structure and how they form have remained poorly understood. Using advanced magic angle spinning solid-state NMR, we directly probe the structure of the rigid core that is at the heart of huntingtin exon1 fibrils and other polyglutamine aggregates, via measurements of long-range intramolecular and intermolecular contacts, backbone and side-chain torsion angles, relaxation measurements, and calculations of chemical shifts. These experiments reveal the presence of β-hairpin-containing β-sheets that are connected through interdigitating extended side chains. Despite dramatic differences in aggregation behavior, huntingtin exon1 fibrils and other polyglutamine-based aggregates contain identical β-strand-based cores. Prior structural models, derived from X-ray fiber diffraction and computational analyses, are shown to be inconsistent with the solid-state NMR results. Internally, the polyglutamine amyloid fibrils are coassembled from differently structured monomers, which we describe as a type of "intrinsic" polymorphism. A stochastic polyglutamine-specific aggregation mechanism is introduced to explain this phenomenon. We show that the aggregation of mutant huntingtin exon1 proceeds via an intramolecular collapse of the expanded polyglutamine domain and discuss the implications of this observation for our understanding of its misfolding and aggregation mechanisms.solid-state NMR | Huntington's disease | amyloid disease | protein aggregation | amyloid

In Huntington’s disease, expansion of a polyglutamine (polyQ) domain in the huntingtin (htt) protein leads to misfolding and aggregation. There is much interest in the molecular features that distinguish monomeric, oligomeric, and fibrillar species that populate the aggregation pathway and likely differ in cytotoxicity. The mechanism and rate of aggregation are greatly affected by the domains flanking the polyQ segment within exon 1 of htt. A “protective” C-terminal proline-rich flanking domain inhibits aggregation by inducing polyproline II structure (PPII) within an extended portion of polyQ. The N-terminal flanking segment (httNT) adopts an α-helical structure as it drives aggregation, helps stabilize oligomers and fibrils, and is seemingly integral to their supramolecular assembly. Via solid-state nuclear magnetic resonance (ssNMR), we probe how, in the mature fibrils, the htt flanking domains impact the polyQ domain and in particular the localization of the β-structured amyloid core. Using residue-specific and uniformly labeled samples, we find that the amyloid core occupies most of the polyQ domain but ends just prior to the prolines. We probe the structural and dynamical features of the remarkably abrupt β-sheet to PPII transition and discuss the potential connections to certain htt-binding proteins. We also examine the httNT α-helix outside the polyQ amyloid core. Despite its presumed structural and demonstrated stabilizing roles in the fibrils, quantitative ssNMR measurements of residue-specific dynamics show that it undergoes distinct solvent-coupled motion. This dynamical feature seems reminiscent of molten-globule-like α-helix-rich features attributed to the nonfibrillar oligomeric species of various amyloidogenic proteins.

The conformational preferences of polyglutamine (polyQ) sequences are of major interest because of their central importance in the expanded CAG repeat diseases that include Huntington’s disease (HD). Here we explore the response of various biophysical parameters to the introduction of β-hairpin motifs within polyQ sequences. These motifs (trpzip, disulfide, D-Pro-Gly, Coulombic attraction, L-Pro-Gly) enhance formation rates and stabilities of amyloid fibrils with degrees of effectiveness well-correlated with their known abilities to enhance β-hairpin formation in other peptides. These changes led to decreases in the critical nucleus for amyloid formation from a value of n* = 4 for a simple, unbroken Q23 sequence to approximate unitary n* values for similar length polyQs containing β-hairpin motifs. At the same time, the morphologies, secondary structures, and bioactivities of the resulting fibrils were essentially unchanged from simple polyQ aggregates. In particular, the signature pattern of SSNMR 13C Gln resonances that appears to be unique to polyQ amyloid is replicated exactly in fibrils from a β-hairpin polyQ. Importantly, while β-hairpin motifs do produce enhancements in the equilibrium constant for nucleation in aggregation reactions, these Kn* values remain quite low (~ 10−10) and there is no evidence for significant embellishment of β-structure within the monomer ensemble. The results indicate an important role for β-turns in the nucleation mechanism and structure of polyQ amyloid and have implications for the nature of the toxic species in expanded CAG repeat diseases.

Excess accumulation of phenylalanine is the characteristic of untreated Phenylketonuria (PKU), a well-known genetic abnormality, which triggers several neurological, physical and developmental severities. However, the fundamental mechanism behind the origin of such diverse health problems, particularly the issue of how they are related to the build-up of phenylalanine molecules in the body, is largely unknown. Here, we show cross-seeding ability of phenylalanine fibrils that can effectively initiate an aggregation process in proteins under physiological conditions, converting native protein structures to β-sheet assembly. The resultant fibrils were found to cause severe hemolysis, yielding a plethora of deformed erythrocytes that is highly relevant to phenylketonuria. Unique arrangement of zwitterionic phenylalanine molecules in their amyloid-like higher order entities is predicted to promote both hydrophobic and electrostatic interaction, sufficient enough to trap proteins and to preferentially interact with the membrane components of RBCs. Since the prevalence of hemolysis and amyloid related psychoneurological severities are mostly observed in PKU patients, we propose that the inherent property of phenylalanine fibrils to trigger hemolysis and to induce protein aggregation may have direct relevance to the disease mechanism of PKU.

Interest in self-association of peptides and proteins is motivated by an interest in the mechanism of physiologically higher order assembly of proteins such as collagen as well as the mechanism of pathological aggregation such as ␤-amyloid formation. The triple helical form of (Pro-Hyp-Gly) 10 , a peptide that has proved a useful model for molecular features of collagen, was found to self-associate, and its association properties are reported here. Turbidity experiments indicate that the triple helical peptide self-assembles at neutral pH via a nucleationgrowth mechanism, with a critical concentration near 1 mM. The associated form is more stable than individual molecules by about 25°C, and the association is reversible. The rate of self-association increases with temperature, supporting an entropically favored process. After self-association, (ProHyp-Gly) 10 forms branched filamentous structures, in contrast with the highly ordered axially periodic structure of collagen fibrils. Yet a number of characteristics of triple helix assembly for the peptide resemble those of collagen fibril formation. These include promotion of fibril formation by neutral pH and increasing temperature; inhibition by sugars; and a requirement for hydroxyproline. It is suggested that these similar features for peptide and collagen self-association are based on common lateral underlying interactions between triple helical molecules mediated by hydrogen-bonded hydration networks involving hydroxyproline.There is increasing interest in the ability of proteins and peptides to self-associate into aggregates, both in normal and pathological processes. Normal self-association processes include fibril formation of collagen and polymerization of actin (1, 2), whereas pathological aggregation of amyloid peptides, ␣-synuclein, and prions is implicated in neurodegenerative diseases (3,4). Interest has focused on the nature of protein aggregation and the molecular and environmental determinants of the self-association process. The study of the ability of collagen-like peptides to aggregate offers an opportunity to characterize a unique system, which may relate to the physiological self-association of collagen molecules.Collagen, the major structural protein in the extracellular matrix, has a characteristic triple helical conformation, consisting of three polyproline II-like chains that are supercoiled around a common axis (5-7). The close packing of the three chains near the central axis generates a requirement for Gly as every third residue, (Gly-X-Y) n , whereas the high content of imino acids Pro and hydroxyproline (Hyp) stabilizes the individual polyproline II-like helices. Although imino acids are highly favorable for the triple helix, the post-translational modification of Pro to Hyp in the Y position confers an additional stabilizing contribution. This further stabilization of Hyp is likely to result from steroelectronic promotion of the more favorable exo ring pucker for the Y position and Hyp involvement in solvent-mediated hydrogen bonding...

Here, we show that aromatic amino acid tyrosine, under a physiologically mimicking condition, readily forms amyloid-like entities that can effectively drive aggregation of different globular proteins and aromatic residues. Tyrosine self-assembly resulted in the formation of cross-β rich regular fibrils as well as spheroidal oligomers. Computational data suggest intermolecular interaction between specifically oriented tyrosine molecules mediated through π-π stacking and H-bonding interactions, mimicking a cross-β-like architecture. Both individual protein samples and mixed protein samples underwent aggregation in the presence of tyrosine fibrils, confirming the occurrence of amyloid cross-seeding. The surface of the tyrosine's amyloid like entities was predicted to trap native protein structures, preferably through hydrophobic and electrostatic interactions initiating an aggregation event. Because tyrosine is a precursor to vital neuromodulators, the inherent cross-seeding potential of the tyrosine fibrils may have direct relevance to amyloid-linked pathologies.

Peptides have been an integral part of the collagen triple‐helix structure story, and have continued to serve as useful models for biophysical studies and for establishing biologically important sequence‐structure‐function relationships. High resolution structures of triple‐helical peptides have confirmed the basic Ramachandran triple‐helix model and provided new insights into the hydration, hydrogen bonding, and sequence dependent helical parameters in collagen. The dependence of collagen triple‐helix stability on the residues in its (Gly‐X‐Y)n repeating sequence has been investigated by measuring melting temperatures of host‐guest peptides and an on‐line collagen stability calculator is now available. Although the presence of Gly as every third residue is essential for an undistorted structure, interruptions in the repeating (Gly‐X‐Y)n amino acid sequence pattern are found in the triple‐helical domains of all nonfibrillar collagens, and are likely to play a role in collagen binding and degradation. Peptide models indicate that small interruptions can be incorporated into a rod‐like triple‐helix with a highly localized effect, which perturbs hydrogen bonds and places the standard triple‐helices on both ends out of register. In contrast to natural interruptions, missense mutations which replace one Gly in a triple‐helix domain by a larger residue have pathological consequences, and studies on peptides containing such Gly substitutions clarify their effect on conformation, stability, and folding. Recent studies suggest peptides may also be useful in defining the basic principles of collagen self‐association to the supramolecular structures found in tissues. © 2008 Wiley Periodicals, Inc. Biopolymers 89: 345–353, 2008.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com