Figure 5. CSPs caused by UbcH5a in ¹⁵N-RNF125^{start31/stop129}.

(A) Overlay of ¹H-¹⁵N HSQC spectra of 0.2 mM ¹⁵N-RNF125^{start31/stop129} collected at 600 MHz before (black), and after addition of 2 molar equivalents of UbcH5a (red). Specific peaks shown in panel B are indicated by arrows. (B) Expanded regions of ¹H-¹⁵N HSQC spectra showing specific residues in the RING domain, C2HC ZnF and Li2^120-128, respectively. Overlays are shown of 0.2 mM ¹⁵N-RNF125^{start31/stop129} before (black), or after UbcH5a was added at a molar ratio of 0.25 (blue), 0.5 (grey), 1 (pink) or 2 (red). Some peaks (V39, L41, R113) broadened beyond detection at the higher molar ratios of UbcH5a. (C) A plot of the combined ¹H and ¹⁵N chemical shift perturbations for ¹⁵N-RNF125^{start31/stop129} residues obtained at 2 molar equivalents of UbcH5a (see A). Assigned residues are in black in the sequence underneath the graph, Pro residues are indicated by (*). ^#indicate T33 and D88 that were too broad in our spectra to be included in the CSP calculation. Red bars represent residues that broadened beyond detection in the presence of UbcH5a. Diagrams of secondary structure elements (α-helices as cylinders, and β-sheets as arrows) and the RNF125^stop129 domain organization are also shown.