10 research outputs found
Summary of effects of SDS on ataxin-3 aggregation.
<p>Schematic summarizing the effects of micellar and non-micellar SDS on both stages of ataxin-3 aggregation.</p
Native Page analysis of M and Z α<sub>1</sub>AT under HDX conditions.
<p>M and Z α<sub>1</sub>AT were incubated in D<sub>2</sub>O buffered with 10 mM Tris (pD 8) at 25°C for up to 2500 seconds. Samples of the proteins were then analyzed by 10% Native PAGE. (A): M α<sub>1</sub>AT t = 0 seconds; (B) M α<sub>1</sub>AT t = 2500 seconds; (C) Z α<sub>1</sub>AT t = 0 seconds; (D) Z α<sub>1</sub>AT t = 2500 seconds and (E) Z α<sub>1</sub>AT polymers purified directly from <i>P. Pastoris</i> 10].</p
Summary of differences in HDX between M and Z α<sub>1</sub>AT.
<p>The structure of α<sub>1</sub>AT (PDB: 1QLP) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102617#pone.0102617-Elliott1" target="_blank">[24]</a> with Red spheres: regions showing increased HDX in Z. Dark blue: regions showing decreased HDX in Z. Cyan: regions showing no significant difference between M and Z. Grey: regions for which no peptides were analyzed.</p
Details of the peptides derived from pepsin digestion and tandem mass spectrometry experiments.
<p>The relative masses used in this study were determined using Sequest.</p><p>Details of the peptides derived from pepsin digestion and tandem mass spectrometry experiments.</p
Aggregation of ataxin-3 in the presence of SDS monitored by SDS-insolubility.
<p>Formation of SDS-insoluble fibrils was followed by taking aliquots from a 30 µM ataxin-3(Q64) timecourse assay at 37°C, pH 7.4. (A) A representative filter-trap membrane of ataxin-3(Q64) with 0–10 mM SDS is shown. QBP1 was added to a ataxin-3(Q64) containing 5 mM SDS as indicated. (B) Analysis of the filter trap membrane by densitometry. Ataxin-3(Q64) is shown with the addition of 0 mM SDS(-▪-), 1 mM SDS(-▴-), 5 mM SDS (-♦- ), 5 mM SDS with QBP1 (-◊-) and 10 mM SDS (-•-). Results from three independent experiments were fit to an exponential curve.</p
Far-UV CD spectra of ataxin-3 variants in increasing concentrations of SDS.
<p>The far-UV CD spectra for (a) ataxin-3(Q64), (b) ataxin-3(Q15) and (c) Josephin were measured at 37°C with increasing concentrations of SDS; 0 mM SDS (<i>black solid line</i>), 1 mM SDS (<i>black dotted line</i>), 5 mM SDS (<i>grey solid line)</i> or 10 mM SDS (<i>grey dashed line</i>). The final protein concentration was 30 µM and the spectra measured with a path length of 0.1 mm.</p
Regions in α<sub>1</sub>AT that are affected by the Z mutation.
<p>(A) Peptides that displayed decreased exchange in Z α<sub>1</sub>AT (Red) compared to M α<sub>1</sub>AT (Black); (B) Peptides that displayed enhanced exchange in Z α<sub>1</sub>AT (Red) compared to M α<sub>1</sub>AT (Black) at 10 sec. (C) The structure of α<sub>1</sub>AT (PDB: 1QLP) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102617#pone.0102617-Elliott1" target="_blank">[24]</a> indicating residues with an increased D<sub>2</sub>O uptake in Z (red) and decreased D<sub>2</sub>O uptake in Z (Blue) after 10 seconds of incubation in D<sub>2</sub>O.</p
Peptic fragments derived from α<sub>1</sub>AT that show comparable exchange kinetics in M and Z α<sub>1</sub>AT.
<p>(A) The kinetics of deuterium incorporation into M α<sub>1</sub>AT (black) and Z α<sub>1</sub>AT (red) by individual peptic fragments which show comparable exchange are shown. The individual data points are the average of three independent experiments for clarity the error bars are not shown. (B) Crystal structure of M α<sub>1</sub>AT (PDB: 1QLP <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102617#pone.0102617-Elliott1" target="_blank">[24]</a>) indicating the location of peptic fragments with comparable exchange highlighted in red.</p
Differences in hydrogen exchange at 2000 seconds between M and Z α<sub>1</sub>AT.
<p>The amino acid sequence of α<sub>1</sub>AT is shown with secondary structure highlighted above the sequence. The 18 peptides used in the study are noted, as double headed arrows. The peptides for both M and Z α<sub>1</sub>AT are colored according to the percentage deuterium incorporation at 2000 seconds: class 1 80–100% (red), class 2 30–80% (yellow) and class 3 0–30% (blue).</p