Skip to main content
. 2018 May 1;7:e36258. doi: 10.7554/eLife.36258

Figure 2. Domain structures of dASCIZ.

(a) Domain structure of dASCIZ, showing the dZnF domain (red) and 7 LC8 binding motifs in its C-terminal domain (blue). Dark blue bars indicate predicted TQT motifs and gray bars indicate the TMT motif (QT3) identified in this study. (b) Sedimentation velocity analysis of the dZnF domain (red), collected at 10°C, and the dLBD (blue), collected at 25°C. (c) Far UV CD spectrum of the ZnF domain (red squares) and the dLBD (blue circles), both collected at 10°C. (d) [15N−1H]-BEST-TROSY spectrum at 850 MHz showing backbone assignments for 133 non-proline residues. Unassigned peaks correspond to the two additional N-terminal residues from the expression vector. The spectrum was recorded at 10°C. (e) A plot of secondary chemical shift differences versus residue number. ΔCα and ΔCβ values were calculated by subtracting the random coil chemical shifts (Tamiola et al., 2010) from the experimentally determined. ΔCα − ΔCβ values < ±1.0 ppm are considered not significant. (f) Plots of R2/R1 and (g) heteronuclear NOE values measured at 10°C indicate high level of disorder. A dotted line is placed at the average value to aid in visualization. Segments corresponding to LC8 recognition motifs, QT1, QT2, QT3, QT4, QT5, QT6, and QT7 are shown. Additional structural characterization of the dLBD by circular dichroism and gel filtration chromatography is shown in Figure 2—figure supplement 1.

Figure 2—source data 1. Tables containing the NMR data that were used to generate the graphs in Figure 2e–g.
Cα and Cβ chemical shifts are listed for Figure 2e, R1 and R2 relaxation rates for Figure 2f, and Iunsat/Isat values for Figure 2g.
elife-36258-fig2-data1.docx (160.6KB, docx)
DOI: 10.7554/eLife.36258.007

Figure 2.

Figure 2—figure supplement 1. dLBD C-terminus is transiently compact.

Figure 2—figure supplement 1.

(a) Far UV CD spectrum of dLBD constructs collected at 10°C: QT1-3 (red), QT2-4 (orange), QT4-6 (green), and QT4-7 (magenta). (b) Size exclusion chromatography of each construct from (a) depicts differences in migration time, using the same color scheme. Size exclusion chromatography was performed on a Superdex 75 10/300 column.