R all proteins and results in a highly amyloidogenic species. In addition, 1 mM SDS Title Loaded From File alsoFigure 1. Far-UV CD spectra of ataxin-3 variants in increasing concentrations of SDS. The far-UV CD spectra for (a) ataxin-3(Q64), (b) ataxin-3(Q15) and (c) Josephin were measured at 37uC with increasing concentrations of SDS; 0 mM SDS (black solid line), 1 mM SDS (black dotted line), 5 mM SDS (grey solid line) or 10 mM SDS (grey dashed line). The final protein concentration was 30 mM and the spectra measured with a path length of 0.1 mm. doi:10.1371/journal.pone.0069416.gresulted in hyperfluorescence of thioT (Fig. 2) which may be related to a greater number of short fibrils being formed. In contrast, at both 5 mM and 10 mM SDS, there is no increase in thioT fluorescence for any of the ataxin-3 variants, thus suggesting that fibril formation is Title Loaded From File suppressed at these micellar SDS concentrations. These results, in which a specific range 16574785 of SDS concentrations around the CMC modulate thioT detectedAggregation of Ataxin-3 in SDSTable 1. Percentage of a-helical content of monomeric protein with SDS present.[SDS] mMAtaxin-3(64) a- helix Standard Error 2.3 2.7 1.9 1.Ataxin-3(Q15) a- helix 30.3 30.4 37.5 39.1 Standard Error 2.6 2.0 2.0 2.Josephin a- helix 30.8 29.5 36.4 35.4 Standard Error 3.5 1.3 2.4 3.0 1 527.5 28.7 32.0 32.doi:10.1371/journal.pone.0069416.tfibrillogenesis, are consistent with those previously reported for a range of other non-polyQ amyloid proteins [37?9].SDS Modulates the Change to b-sheet Secondary Structure Typical of AggregationWith the intriguing formation of thioT unreactive fibrils by 5 mM SDS, we then went on to characterize the changes in secondary structure occurring during aggregation. SDS induces an increase in a-helical structure at concentrations above the CMC (Fig. 1), however a key event in fibrillogenesis is the gain of b-sheet structure, and hence far-UV CD was used to follow the impact of SDS upon this structural transition. As previously reported, we observed that in the absence of SDS ataxin-3(Q64) converts to a b-sheet rich fibrillar species (Fig. 4A). The loss in signal observed over time has been previously suggested to reflect an increase in light scatter [9]. Incubation in 1 mM SDS (Fig. 4B) accelerates the kinetics of aggregation such that by four hours there has been substantial loss of helical structure and a conversion to b-sheet structure which continues over time with a loss of signal similar to that seen in the absence of SDS at 100 hours (Fig. 4A). Incubation of ataxin-3(Q64) in both 5 mM and 10 mM SDS leads to a retention of a-helical structure over 100 hours, and for 10 mM SDS there is a small increase in the minima at 208 nm and 222 nm (Fig. 4D). This is consistent with the lack of aggregation detected with 10 mM SDS throughout this study and suggests that SDS has stabilized the a-helical structure to the extent that the conversion to b-sheet is prevented. With 5 mM SDS present, the retention of a-helical structure over time concurs with the lack of thioT fluorescence observed (Fig. 2) and thus suggests that the SDS-insoluble fibrils being formed (Fig. 3A) are more similar to 23977191 amorphous aggregates than the b-sheet rich amyloid-like fibrils typically formed by ataxin-3. Interestingly, these aggregates are still formed via interactions of the polyQ tract, as addition of QBP1 inhibits their formation (Fig. 3A). The same effects of SDS on the change in secondary structure over time were also observed fo.R all proteins and results in a highly amyloidogenic species. In addition, 1 mM SDS alsoFigure 1. Far-UV CD spectra of ataxin-3 variants in increasing concentrations of SDS. The far-UV CD spectra for (a) ataxin-3(Q64), (b) ataxin-3(Q15) and (c) Josephin were measured at 37uC with increasing concentrations of SDS; 0 mM SDS (black solid line), 1 mM SDS (black dotted line), 5 mM SDS (grey solid line) or 10 mM SDS (grey dashed line). The final protein concentration was 30 mM and the spectra measured with a path length of 0.1 mm. doi:10.1371/journal.pone.0069416.gresulted in hyperfluorescence of thioT (Fig. 2) which may be related to a greater number of short fibrils being formed. In contrast, at both 5 mM and 10 mM SDS, there is no increase in thioT fluorescence for any of the ataxin-3 variants, thus suggesting that fibril formation is suppressed at these micellar SDS concentrations. These results, in which a specific range 16574785 of SDS concentrations around the CMC modulate thioT detectedAggregation of Ataxin-3 in SDSTable 1. Percentage of a-helical content of monomeric protein with SDS present.[SDS] mMAtaxin-3(64) a- helix Standard Error 2.3 2.7 1.9 1.Ataxin-3(Q15) a- helix 30.3 30.4 37.5 39.1 Standard Error 2.6 2.0 2.0 2.Josephin a- helix 30.8 29.5 36.4 35.4 Standard Error 3.5 1.3 2.4 3.0 1 527.5 28.7 32.0 32.doi:10.1371/journal.pone.0069416.tfibrillogenesis, are consistent with those previously reported for a range of other non-polyQ amyloid proteins [37?9].SDS Modulates the Change to b-sheet Secondary Structure Typical of AggregationWith the intriguing formation of thioT unreactive fibrils by 5 mM SDS, we then went on to characterize the changes in secondary structure occurring during aggregation. SDS induces an increase in a-helical structure at concentrations above the CMC (Fig. 1), however a key event in fibrillogenesis is the gain of b-sheet structure, and hence far-UV CD was used to follow the impact of SDS upon this structural transition. As previously reported, we observed that in the absence of SDS ataxin-3(Q64) converts to a b-sheet rich fibrillar species (Fig. 4A). The loss in signal observed over time has been previously suggested to reflect an increase in light scatter [9]. Incubation in 1 mM SDS (Fig. 4B) accelerates the kinetics of aggregation such that by four hours there has been substantial loss of helical structure and a conversion to b-sheet structure which continues over time with a loss of signal similar to that seen in the absence of SDS at 100 hours (Fig. 4A). Incubation of ataxin-3(Q64) in both 5 mM and 10 mM SDS leads to a retention of a-helical structure over 100 hours, and for 10 mM SDS there is a small increase in the minima at 208 nm and 222 nm (Fig. 4D). This is consistent with the lack of aggregation detected with 10 mM SDS throughout this study and suggests that SDS has stabilized the a-helical structure to the extent that the conversion to b-sheet is prevented. With 5 mM SDS present, the retention of a-helical structure over time concurs with the lack of thioT fluorescence observed (Fig. 2) and thus suggests that the SDS-insoluble fibrils being formed (Fig. 3A) are more similar to 23977191 amorphous aggregates than the b-sheet rich amyloid-like fibrils typically formed by ataxin-3. Interestingly, these aggregates are still formed via interactions of the polyQ tract, as addition of QBP1 inhibits their formation (Fig. 3A). The same effects of SDS on the change in secondary structure over time were also observed fo.
