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Infrared nanospectroscopy characterization of oligomeric and fibrillar aggregates during amyloid formation.

Ruggeri FS, Longo G, Faggiano S, Lipiec E, Pastore A, Dietler G - Nat Commun (2015)

Bottom Line: We describe their secondary structure, monitoring at the nanoscale an α-to-β transition, and couple these studies with an independent measurement of the evolution of their intrinsic stiffness.These results suggest that the aggregation of Josephin proceeds from the monomer state to the formation of spheroidal intermediates with a native structure.Only successively, these intermediates evolve into misfolded aggregates and into the final fibrils.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Physique de la Matière Vivante, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland.

ABSTRACT
Amyloids are insoluble protein fibrillar aggregates. The importance of characterizing their aggregation has steadily increased because of their link to human diseases and material science applications. In particular, misfolding and aggregation of the Josephin domain of ataxin-3 is implicated in spinocerebellar ataxia-3. Infrared nanospectroscopy, simultaneously exploiting atomic force microscopy and infrared spectroscopy, can characterize at the nanoscale the conformational rearrangements of proteins during their aggregation. Here we demonstrate that we can individually characterize the oligomeric and fibrillar species formed along the amyloid aggregation. We describe their secondary structure, monitoring at the nanoscale an α-to-β transition, and couple these studies with an independent measurement of the evolution of their intrinsic stiffness. These results suggest that the aggregation of Josephin proceeds from the monomer state to the formation of spheroidal intermediates with a native structure. Only successively, these intermediates evolve into misfolded aggregates and into the final fibrils.

No MeSH data available.


Related in: MedlinePlus

PCA analysis.The results of PCA analysis in the spectral range 1,800–1,270 cm−1 (excluding 1,620–1,480 cm−1) applied to three groups of spectra: native oligomers, misfolded oligomers and fibrils. (a) Three-dimensional scores plot. (b) Loadings plot. The arrows indicate the fingerprint of the conformational changes during amyloid formation.
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f6: PCA analysis.The results of PCA analysis in the spectral range 1,800–1,270 cm−1 (excluding 1,620–1,480 cm−1) applied to three groups of spectra: native oligomers, misfolded oligomers and fibrils. (a) Three-dimensional scores plot. (b) Loadings plot. The arrows indicate the fingerprint of the conformational changes during amyloid formation.

Mentions: The collected spectra were placed in the space of new independent variables, the PCs. Thus, we could distinguish three distinct clusters, corresponding to three groups of aggregates: native oligomers, misfolded oligomers and amyloid fibrils (Fig. 6a). PC-1, which explains 50% of total variance within the ensemble of spectra (black in Fig. 5b), was positively correlated with the wavenumbers attributed to the amide I in spectral range from 1,710 to 1,680 cm−1, to the COO− vibration around 1,430 cm−1 and to the amide III at 1,285 cm−1 (Fig. 6b). The scores plot showed that these three bands were positively correlated with the PC-1 scores for fibrils and, partially, for misfolded oligomers and negatively correlated with the PC-1 scores for native oligomers and partially for misfolded oligomers (Fig. 6a). This showed that the amide I band between 1,655–1,620 cm−1 and the amide III band at 1,308 cm−1 are typical of these species. The PC-3, representing 10% of the total variance, clearly indicated a shift of the COO− band. The PC-3 was positively correlated with the COO− band at 1,430 cm−1 (fibrils and partially misfolded oligomers) and negatively correlated with the band at 1,412 cm−1 (native oligomers and partially misfolded oligomers). We also tested PCA on the second derivatives of the spectra (Supplementary Fig. 7). The conformational change described by the spectra was in good agreement with previous studies in literature using bulk infrared spectroscopy3132. During aggregation, the Fourier transform infrared spectroscopy spectrum changed as a function of time, and the main modifications to the amide I band signal were the decrease in random coil and α-helical structure (1,655 cm−1), the increase in antiparallel β-sheet content (1,695 cm−1) and the decrease in the native β-sheet content. The results achieved using PCA on second derivatives were well in agreement with the analysis of the raw spectra (Supplementary Discussion and Supplementary Fig. 8).


Infrared nanospectroscopy characterization of oligomeric and fibrillar aggregates during amyloid formation.

Ruggeri FS, Longo G, Faggiano S, Lipiec E, Pastore A, Dietler G - Nat Commun (2015)

PCA analysis.The results of PCA analysis in the spectral range 1,800–1,270 cm−1 (excluding 1,620–1,480 cm−1) applied to three groups of spectra: native oligomers, misfolded oligomers and fibrils. (a) Three-dimensional scores plot. (b) Loadings plot. The arrows indicate the fingerprint of the conformational changes during amyloid formation.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4525161&req=5

f6: PCA analysis.The results of PCA analysis in the spectral range 1,800–1,270 cm−1 (excluding 1,620–1,480 cm−1) applied to three groups of spectra: native oligomers, misfolded oligomers and fibrils. (a) Three-dimensional scores plot. (b) Loadings plot. The arrows indicate the fingerprint of the conformational changes during amyloid formation.
Mentions: The collected spectra were placed in the space of new independent variables, the PCs. Thus, we could distinguish three distinct clusters, corresponding to three groups of aggregates: native oligomers, misfolded oligomers and amyloid fibrils (Fig. 6a). PC-1, which explains 50% of total variance within the ensemble of spectra (black in Fig. 5b), was positively correlated with the wavenumbers attributed to the amide I in spectral range from 1,710 to 1,680 cm−1, to the COO− vibration around 1,430 cm−1 and to the amide III at 1,285 cm−1 (Fig. 6b). The scores plot showed that these three bands were positively correlated with the PC-1 scores for fibrils and, partially, for misfolded oligomers and negatively correlated with the PC-1 scores for native oligomers and partially for misfolded oligomers (Fig. 6a). This showed that the amide I band between 1,655–1,620 cm−1 and the amide III band at 1,308 cm−1 are typical of these species. The PC-3, representing 10% of the total variance, clearly indicated a shift of the COO− band. The PC-3 was positively correlated with the COO− band at 1,430 cm−1 (fibrils and partially misfolded oligomers) and negatively correlated with the band at 1,412 cm−1 (native oligomers and partially misfolded oligomers). We also tested PCA on the second derivatives of the spectra (Supplementary Fig. 7). The conformational change described by the spectra was in good agreement with previous studies in literature using bulk infrared spectroscopy3132. During aggregation, the Fourier transform infrared spectroscopy spectrum changed as a function of time, and the main modifications to the amide I band signal were the decrease in random coil and α-helical structure (1,655 cm−1), the increase in antiparallel β-sheet content (1,695 cm−1) and the decrease in the native β-sheet content. The results achieved using PCA on second derivatives were well in agreement with the analysis of the raw spectra (Supplementary Discussion and Supplementary Fig. 8).

Bottom Line: We describe their secondary structure, monitoring at the nanoscale an α-to-β transition, and couple these studies with an independent measurement of the evolution of their intrinsic stiffness.These results suggest that the aggregation of Josephin proceeds from the monomer state to the formation of spheroidal intermediates with a native structure.Only successively, these intermediates evolve into misfolded aggregates and into the final fibrils.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire de Physique de la Matière Vivante, Ecole Polytechnique Fédérale de Lausanne (EPFL), CH-1015 Lausanne, Switzerland.

ABSTRACT
Amyloids are insoluble protein fibrillar aggregates. The importance of characterizing their aggregation has steadily increased because of their link to human diseases and material science applications. In particular, misfolding and aggregation of the Josephin domain of ataxin-3 is implicated in spinocerebellar ataxia-3. Infrared nanospectroscopy, simultaneously exploiting atomic force microscopy and infrared spectroscopy, can characterize at the nanoscale the conformational rearrangements of proteins during their aggregation. Here we demonstrate that we can individually characterize the oligomeric and fibrillar species formed along the amyloid aggregation. We describe their secondary structure, monitoring at the nanoscale an α-to-β transition, and couple these studies with an independent measurement of the evolution of their intrinsic stiffness. These results suggest that the aggregation of Josephin proceeds from the monomer state to the formation of spheroidal intermediates with a native structure. Only successively, these intermediates evolve into misfolded aggregates and into the final fibrils.

No MeSH data available.


Related in: MedlinePlus