Observation of Oligomeric States Indicates a High Structural Flexibility Required for the Onset of Polyglutamine Fibrillization.

Polyglutamine (polyQ) diseases are caused by misfolding and aggregation of expanded polyQ tracts in the affected protein. PolyQ fibrils have been studied in detail; however, less is known about oligomeric precursor states. By a combination of time-resolved temperature-jump (T-jump) infrared (IR) spectroscopy and an appropriately tailored polyQ model peptide, we succeeded in disentangling conformational dynamics in the heterogeneous ensemble of states evolving during aggregation. Individual structural elements could be differentiated by IR-specific signatures, i.e., hairpin monomers, ß-structured oligomers, and disordered structure. Submillisecond dynamics were observed for early oligomeric states in contrast to the slow dynamics of fibril growth. We propose that a high structural flexibility of oligomers is required to initiate fibril formation, but not after a fibrillar structure has consolidated and the fibril just grows. Our study reveals that structural flexibility changes at different stages in the aggregation process, from fibril initiation to fibril growth.

PolyQ aggregation studied by model peptides with intrinsic tryptophan fluorophores.

Polyglutamine (polyQ) model peptides are ideally suited to analyze the involvement of glutamines in the disease-related aggregation onset. Here we use a template-assisted design of polyQ-rich hairpin peptides (Trpzip-Qn) to monitor structural stability with fluorescence spectroscopy. The hairpin model imitates the monomeric motif of a polyQ fibril and is stabilized by hydrophobic interactions of two cross-strand pairs of tryptophans (Trps) which are used as fluorophores to report on structural changes. The Trps also frame the polyQ repeats located on each hairpin strand with a different number of glutamines (Qn). Single-stranded sequences mimic the unfolded state and were used as references to differentiate the intrinsic fluorescence signal from the spectral effect caused by structural changes. Temperature-induced hairpin unfolding was monitored by the spectral shift of the Trp fluorescence signal and transition temperatures were determined. The magnitude of the spectral shift indicates the degree of structural disorder. We observed that a longer polyQ repeat is more disordered and weakens the cross-strand Trp-Trp interactions resulting in a decrease of the spectral shift. Aggregation to a fibrillar and more ordered structure shows an increase of the spectral shift. In addition, a band at 280 nm occurs in the spectrum which clearly correlates with the turbidity of the sample and is attributed to scattering of larger aggregated structures. Our study reveals that the number of glutamines, pH and temperature affect structural stability and aggregation of polyQ repeats.

Template-assisted design of monomeric polyQ models to unravel the unique role of glutamine side chains in disease-related aggregation.

Expanded polyglutamine (polyQ) sequences cause numerous neurodegenerative diseases which are accompanied by the formation of polyQ fibrils. The unique role of glutamines in the aggregation onset is undoubtedly accepted and a lot structural data of the fibrils have been acquired, however side-chain specific structural dynamics inducing oligomerization are not well understood yet. To analyze spectroscopically the nucleation process, we designed various template-assisted glutamine-rich ß-hairpin monomers mimicking the structural motif of a polyQ fibril. In a top-down strategy, we use a template which forms a well-defined stable hairpin in solution, insert polyQ-rich sequences into each strand and monitor the effects of individual glutamines by NMR, CD and IR spectroscopic approaches. The design was further advanced by alternating glutamines with other amino acids (T, W, E, K), thereby enhancing the solubility and increasing the number of cross-strand interacting glutamine side chains. Our spectroscopic studies reveal a decreasing hairpin stability with increased glutamine content and demonstrate the enormous impact of only a few glutamines - far below the disease threshold - to destabilize structure. Furthermore, we could access sub-ms conformational dynamics of monomeric polyQ-rich peptides by laser-excited temperature-jump IR spectroscopy. Both, the increased number of interacting glutamines and higher concentrations are key parameters to induce oligomerization. Concentration-dependent time-resolved IR measurements indicate an additional slower kinetic phase upon oligomer formation. The here presented peptide models enable spectroscopic molecular analyses to distinguish between monomer and oligomer dynamics in the early steps of polyQ fibril formation and in a side-chain specific manner.