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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

AFM-infrared chemical maps and spectra of Josephin proteins before incubation at 37 °C.(a) AFM height image. Infrared absorption map at (b) 1,700 cm−1 (amide I), (c) 1,655 cm−1 (amide I), (d) 1,300 cm−1 (amide III). Scale bar, 2 μm. (e) Infrared spectra. (f) Average oligomeric infrared spectrum and secondary-structure deconvolution of amide I band.
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f3: AFM-infrared chemical maps and spectra of Josephin proteins before incubation at 37 °C.(a) AFM height image. Infrared absorption map at (b) 1,700 cm−1 (amide I), (c) 1,655 cm−1 (amide I), (d) 1,300 cm−1 (amide III). Scale bar, 2 μm. (e) Infrared spectra. (f) Average oligomeric infrared spectrum and secondary-structure deconvolution of amide I band.

Mentions: We measured the structural properties of Josephin before incubation at 37 °C (Fig. 3). The images show the morphology (Fig. 3a) and the absorption of infrared light in the amide I (1,700 and 1,655 cm−1, Fig. 3b,c) and amide III (1,300 cm−1, Fig. 3d) bands. From the comparison of the absorption maps and considering the components of amide I band related to proteins' secondary structure (Supplementary Discussion), we can infer that the oligomers adsorb mainly infrared radiation in the spectral region of the amide I band, related to random coil and α-helical conformations (1,655 cm−1). These chemical absorption maps clearly indicated that the instrument could detect aggregates as small as 50 nm in average height. This height corresponds, for a hemispherical structure on a surface, to a maximum height of ∼100 nm (Supplementary Discussion and Supplementary Figs 3–4). To confirm quantitatively the trend of absorption shown by infrared, we acquired spectra of individual oligomeric aggregates present on the surface (Fig. 3e). This was achieved by positioning the AFM tip on each structure and collecting several spectra inside their area (minimum 10). The comparison of these spectra showed that they had similar peak amplitudes and positions. The amide I band was approximately within 1,655 cm−1 (α-helix) and 1,620 cm−1 (β-sheet) and centred at ∼1,630–1,640 cm−1 (random coil) with a shoulder within 1,720–1,680 cm−1 (antiparallel β-sheets, β-turns and side-chain vibrations). The position of the amide II band was approximately within 1,590–1,560 cm−1. However, it was not easily measurable because of low signal-to-noise ratio in this spectral range (Supplementary Discussion and Supplementary Figs 4 and5). The amide III band, centred at 1,300 cm−1, appeared to be coupled with a second peak, which is visible at 1,410–1,400 cm−1. This was already observed in our studies on lysozyme, where we highlighted a change of the relative ratio of the amplitude of this peak and the amide III band during the process of aggregation23. This peak can be attributed to a combination of COO−, C–N, C–C stretching vibration, C–H and N–H bending, in plane O–H bending. A weaker contribution derives from the vibration of the glutamine (1,410 cm−1), glutamic acid (1,404 cm−1) and aspartic acid (1,402 cm−1) side chains2930.


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)

AFM-infrared chemical maps and spectra of Josephin proteins before incubation at 37 °C.(a) AFM height image. Infrared absorption map at (b) 1,700 cm−1 (amide I), (c) 1,655 cm−1 (amide I), (d) 1,300 cm−1 (amide III). Scale bar, 2 μm. (e) Infrared spectra. (f) Average oligomeric infrared spectrum and secondary-structure deconvolution of amide I band.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: AFM-infrared chemical maps and spectra of Josephin proteins before incubation at 37 °C.(a) AFM height image. Infrared absorption map at (b) 1,700 cm−1 (amide I), (c) 1,655 cm−1 (amide I), (d) 1,300 cm−1 (amide III). Scale bar, 2 μm. (e) Infrared spectra. (f) Average oligomeric infrared spectrum and secondary-structure deconvolution of amide I band.
Mentions: We measured the structural properties of Josephin before incubation at 37 °C (Fig. 3). The images show the morphology (Fig. 3a) and the absorption of infrared light in the amide I (1,700 and 1,655 cm−1, Fig. 3b,c) and amide III (1,300 cm−1, Fig. 3d) bands. From the comparison of the absorption maps and considering the components of amide I band related to proteins' secondary structure (Supplementary Discussion), we can infer that the oligomers adsorb mainly infrared radiation in the spectral region of the amide I band, related to random coil and α-helical conformations (1,655 cm−1). These chemical absorption maps clearly indicated that the instrument could detect aggregates as small as 50 nm in average height. This height corresponds, for a hemispherical structure on a surface, to a maximum height of ∼100 nm (Supplementary Discussion and Supplementary Figs 3–4). To confirm quantitatively the trend of absorption shown by infrared, we acquired spectra of individual oligomeric aggregates present on the surface (Fig. 3e). This was achieved by positioning the AFM tip on each structure and collecting several spectra inside their area (minimum 10). The comparison of these spectra showed that they had similar peak amplitudes and positions. The amide I band was approximately within 1,655 cm−1 (α-helix) and 1,620 cm−1 (β-sheet) and centred at ∼1,630–1,640 cm−1 (random coil) with a shoulder within 1,720–1,680 cm−1 (antiparallel β-sheets, β-turns and side-chain vibrations). The position of the amide II band was approximately within 1,590–1,560 cm−1. However, it was not easily measurable because of low signal-to-noise ratio in this spectral range (Supplementary Discussion and Supplementary Figs 4 and5). The amide III band, centred at 1,300 cm−1, appeared to be coupled with a second peak, which is visible at 1,410–1,400 cm−1. This was already observed in our studies on lysozyme, where we highlighted a change of the relative ratio of the amplitude of this peak and the amide III band during the process of aggregation23. This peak can be attributed to a combination of COO−, C–N, C–C stretching vibration, C–H and N–H bending, in plane O–H bending. A weaker contribution derives from the vibration of the glutamine (1,410 cm−1), glutamic acid (1,404 cm−1) and aspartic acid (1,402 cm−1) side chains2930.

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