Fig 2. G70N substitution in Ydj1 affects structural properties around the junction of J-domain and Gly-rich region.

(A) Single turnover ATPase assays were performed using ³²P-ATP/Ssa1 complex. Data are presented as fold-stimulation at indicated ratios of Ydj1 or Ydj1G70N, relative to basal Ssa1activity. Error bars indicate the standard deviation. The mean of 6 experiments is plotted. (B) (left) 10-fold serial dilutions of cells lacking Sis1 (sis1-Δ) carrying a plasmid with an insert encoding WT Sis1 (SIS1) or the 109 or 134 residue N-terminal Ydj1 fragment with the G70N substitution (ydj1₁₀₉G70N and ydj1₁₃₄G70N, respectively). (right) Lysates made from these strains were subjected to immunoblot analysis using antibodies specific for the Ydj1 J-domain and, as a control, Ssc1. (C) NMR analysis of conformational changes in Ydj1₁₀₉ due to G70N substitution. (top) Comparison of 2D ¹H,¹⁵N HSQC spectra obtained for Ydj1₁₀₉WT (black) and Ydj1₁₀₉G70N (green). The signal position of each residue is defined by chemical shifts along the ¹⁵N (y-axis) and ¹H (x-axis) dimension, which reflects protein conformation. (right) Overlay of the two HSQC spectra. Most signals are very similar, with a number of signals exhibit substantial differences in position. Arrows indicate signals of the 8 most affected residues: WT (solid), G70N (dotted). (bottom) Histogram representing differences in each residue’s signal position in Ydj1₁₀₉WT and Ydj1₁₀₉G70N HSQC spectra. Change indicated by height of bar, calculated as the combined difference in chemical shifts between signals for each residue. The magnitudes of the chemical shift perturbations (CSP) are color-coded: (red) CSP >0.1 ppm; (gray) CSP ≤ 0.1 ppm; (blue rectangles) signals from residues T5-D9 and L57-Q68 that were identified in the Ydj1₁₀₉WT spectrum, but not in Ydj1₁₀₉G70N; (star) proline residues, which have no signal in HSQC spectra. The α-helices of the J-domain are indicated at the top of the panel. The G70N substitution is indicated by a dotted line.