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A covalent homodimer probing early oligomers along amyloid aggregation.

Halabelian L, Relini A, Barbiroli A, Penco A, Bolognesi M, Ricagno S - Sci Rep (2015)

Bottom Line: In this study, by mutating Ser33 to Cys, and assembling the disulphide-stabilized β2m homodimer (DimC33), such DD strand interface was locked.Three distinct crystal structures of DimC33 suggest that dimerization through the DD interface is instrumental for enhancing DimC33 aggregation propensity.Furthermore, the crystal structure of DimC33 in complex with the amyloid-specific dye Thioflavin-T pinpoints a second interface, which likely participates in the first steps of β2m aggregation.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Bioscienze, Università degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy.

ABSTRACT
Early oligomers are crucial in amyloid aggregation; however, due to their transient nature they are among the least structurally characterized species. We focused on the amyloidogenic protein beta2-microglobulin (β2m) whose early oligomers are still a matter of debate. An intermolecular interaction between D strands of facing β2m molecules was repeatedly observed, suggesting that such interface may be relevant for β2m dimerization. In this study, by mutating Ser33 to Cys, and assembling the disulphide-stabilized β2m homodimer (DimC33), such DD strand interface was locked. Although the isolated DimC33 display a stability similar to wt β2m under native conditions, it shows enhanced amyloid aggregation propensity. Three distinct crystal structures of DimC33 suggest that dimerization through the DD interface is instrumental for enhancing DimC33 aggregation propensity. Furthermore, the crystal structure of DimC33 in complex with the amyloid-specific dye Thioflavin-T pinpoints a second interface, which likely participates in the first steps of β2m aggregation. The present data provide new insight into β2m early steps of amyloid aggregation.

No MeSH data available.


Related in: MedlinePlus

Thermal stability and Far-UV spectra analysis by circular dichroism.(A) Temperature ramps for DimC33 in red and wt β2m in black monitored at 202 nm, temperature slope 50 °C/h. Signals were reported as fractional variation of the total change. The melting temperature (Tm) values for DimC33 and wt β2m are shown in the graph. (B) Far-UV spectra for DimC33 and wt β2m in a buffer containing 100 mM sodium chloride, 50 mM sodium phosphate buffer pH 7.4, recorded under three different conditions: No TFE (black curves); in presence of 10% (v/v) TFE (red curves); and in 20% (v/v) TFE (blue curves).
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f2: Thermal stability and Far-UV spectra analysis by circular dichroism.(A) Temperature ramps for DimC33 in red and wt β2m in black monitored at 202 nm, temperature slope 50 °C/h. Signals were reported as fractional variation of the total change. The melting temperature (Tm) values for DimC33 and wt β2m are shown in the graph. (B) Far-UV spectra for DimC33 and wt β2m in a buffer containing 100 mM sodium chloride, 50 mM sodium phosphate buffer pH 7.4, recorded under three different conditions: No TFE (black curves); in presence of 10% (v/v) TFE (red curves); and in 20% (v/v) TFE (blue curves).

Mentions: The stability of DimC33 in solution was assessed by circular dichroism. Thermal unfolding monitored by Far-UV indicates that, as for wt β2m, DimC33 unfolds according to a simple sigmoidal curve and displays a Tm value close to that of the wt protein (TmDimC33 60.2 ± 0.3 °C; Tmwt 62.4 ± 0.3 °C) (Fig. 2a). Then, Far-UV spectra of DimC33 and of wt β2m in phosphate buffer, in 10% and 20% TFE were collected, showing that native spectra are well superposable and that both proteins display a native secondary structure content in 10% TFE; conversely the β2m fold is perturbed in 20% TFE (Fig. 2b). Thus, the spectroscopic data suggest that the engineered mutation and the achievement of a covalent dimeric state do not alter β2m thermodynamic stability or its overall fold in DimC33.


A covalent homodimer probing early oligomers along amyloid aggregation.

Halabelian L, Relini A, Barbiroli A, Penco A, Bolognesi M, Ricagno S - Sci Rep (2015)

Thermal stability and Far-UV spectra analysis by circular dichroism.(A) Temperature ramps for DimC33 in red and wt β2m in black monitored at 202 nm, temperature slope 50 °C/h. Signals were reported as fractional variation of the total change. The melting temperature (Tm) values for DimC33 and wt β2m are shown in the graph. (B) Far-UV spectra for DimC33 and wt β2m in a buffer containing 100 mM sodium chloride, 50 mM sodium phosphate buffer pH 7.4, recorded under three different conditions: No TFE (black curves); in presence of 10% (v/v) TFE (red curves); and in 20% (v/v) TFE (blue curves).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4588566&req=5

f2: Thermal stability and Far-UV spectra analysis by circular dichroism.(A) Temperature ramps for DimC33 in red and wt β2m in black monitored at 202 nm, temperature slope 50 °C/h. Signals were reported as fractional variation of the total change. The melting temperature (Tm) values for DimC33 and wt β2m are shown in the graph. (B) Far-UV spectra for DimC33 and wt β2m in a buffer containing 100 mM sodium chloride, 50 mM sodium phosphate buffer pH 7.4, recorded under three different conditions: No TFE (black curves); in presence of 10% (v/v) TFE (red curves); and in 20% (v/v) TFE (blue curves).
Mentions: The stability of DimC33 in solution was assessed by circular dichroism. Thermal unfolding monitored by Far-UV indicates that, as for wt β2m, DimC33 unfolds according to a simple sigmoidal curve and displays a Tm value close to that of the wt protein (TmDimC33 60.2 ± 0.3 °C; Tmwt 62.4 ± 0.3 °C) (Fig. 2a). Then, Far-UV spectra of DimC33 and of wt β2m in phosphate buffer, in 10% and 20% TFE were collected, showing that native spectra are well superposable and that both proteins display a native secondary structure content in 10% TFE; conversely the β2m fold is perturbed in 20% TFE (Fig. 2b). Thus, the spectroscopic data suggest that the engineered mutation and the achievement of a covalent dimeric state do not alter β2m thermodynamic stability or its overall fold in DimC33.

Bottom Line: In this study, by mutating Ser33 to Cys, and assembling the disulphide-stabilized β2m homodimer (DimC33), such DD strand interface was locked.Three distinct crystal structures of DimC33 suggest that dimerization through the DD interface is instrumental for enhancing DimC33 aggregation propensity.Furthermore, the crystal structure of DimC33 in complex with the amyloid-specific dye Thioflavin-T pinpoints a second interface, which likely participates in the first steps of β2m aggregation.

View Article: PubMed Central - PubMed

Affiliation: Dipartimento di Bioscienze, Università degli Studi di Milano, Via Celoria 26, 20133 Milan, Italy.

ABSTRACT
Early oligomers are crucial in amyloid aggregation; however, due to their transient nature they are among the least structurally characterized species. We focused on the amyloidogenic protein beta2-microglobulin (β2m) whose early oligomers are still a matter of debate. An intermolecular interaction between D strands of facing β2m molecules was repeatedly observed, suggesting that such interface may be relevant for β2m dimerization. In this study, by mutating Ser33 to Cys, and assembling the disulphide-stabilized β2m homodimer (DimC33), such DD strand interface was locked. Although the isolated DimC33 display a stability similar to wt β2m under native conditions, it shows enhanced amyloid aggregation propensity. Three distinct crystal structures of DimC33 suggest that dimerization through the DD interface is instrumental for enhancing DimC33 aggregation propensity. Furthermore, the crystal structure of DimC33 in complex with the amyloid-specific dye Thioflavin-T pinpoints a second interface, which likely participates in the first steps of β2m aggregation. The present data provide new insight into β2m early steps of amyloid aggregation.

No MeSH data available.


Related in: MedlinePlus