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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

Young's modulus increases as a function of aggregation.AFM quantitative imaging of: (a) oligomeric proteins at 0 day, (b) oligomers after 2 days and (c) fibrillar structures after 7 days of incubation at 37 °C. Scale bar, 2 μm. Stiffness cross-sections of (d) oligomers at 0 day, (e) oligomers at 2 days and (f) fibrillar structures.
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f2: Young's modulus increases as a function of aggregation.AFM quantitative imaging of: (a) oligomeric proteins at 0 day, (b) oligomers after 2 days and (c) fibrillar structures after 7 days of incubation at 37 °C. Scale bar, 2 μm. Stiffness cross-sections of (d) oligomers at 0 day, (e) oligomers at 2 days and (f) fibrillar structures.

Mentions: To map the evolution of the nanomechanical properties of the Josephin aggregates, we used a fast force–volume system (quantitative imaging (QI)). We produced QI maps of the oligomeric (Fig. 2a,b) and fibrillar (Fig. 2c) structures. Before incubation, the spheroidal oligomers had a uniform Young's modulus of 390±170 MPa (Fig. 2d). This value is appreciably smaller than what expected for an amyloid structure182526. After 2 days of incubation, the sample did not have uniform mechanical properties and two different populations were observed. The first showed stiffness similar to the previous time point (470±150 MPa), while the latter showed a larger Young's modulus of 0.95±0.55 GPa (Fig. 2e), a value consistent with formation of a β-rich structure due to misfolding2728. The final fibrillar structures, after 7 days of incubation, had uniform stiffness of 1.70±0.65 GPa (Fig. 2f), in good agreement with the generally accepted value of stiffness of a mature and complete amyloid cross β-sheet structure182526.


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)

Young's modulus increases as a function of aggregation.AFM quantitative imaging of: (a) oligomeric proteins at 0 day, (b) oligomers after 2 days and (c) fibrillar structures after 7 days of incubation at 37 °C. Scale bar, 2 μm. Stiffness cross-sections of (d) oligomers at 0 day, (e) oligomers at 2 days and (f) fibrillar structures.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2: Young's modulus increases as a function of aggregation.AFM quantitative imaging of: (a) oligomeric proteins at 0 day, (b) oligomers after 2 days and (c) fibrillar structures after 7 days of incubation at 37 °C. Scale bar, 2 μm. Stiffness cross-sections of (d) oligomers at 0 day, (e) oligomers at 2 days and (f) fibrillar structures.
Mentions: To map the evolution of the nanomechanical properties of the Josephin aggregates, we used a fast force–volume system (quantitative imaging (QI)). We produced QI maps of the oligomeric (Fig. 2a,b) and fibrillar (Fig. 2c) structures. Before incubation, the spheroidal oligomers had a uniform Young's modulus of 390±170 MPa (Fig. 2d). This value is appreciably smaller than what expected for an amyloid structure182526. After 2 days of incubation, the sample did not have uniform mechanical properties and two different populations were observed. The first showed stiffness similar to the previous time point (470±150 MPa), while the latter showed a larger Young's modulus of 0.95±0.55 GPa (Fig. 2e), a value consistent with formation of a β-rich structure due to misfolding2728. The final fibrillar structures, after 7 days of incubation, had uniform stiffness of 1.70±0.65 GPa (Fig. 2f), in good agreement with the generally accepted value of stiffness of a mature and complete amyloid cross β-sheet structure182526.

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