Fig. 2.

Estimation of the binding affinity of human REV1 (932–1039) for ubiquitin. (a) NMR spectroscopy-based titration of ¹⁵N-labeled REV1 with non-labeled ubiquitin. REV1 was at initial concentration of 0.5 mM. Multiple apparent K_d values were determined from the changes in chemical shifts for five REV1 residues by nonlinear least-squares analysis using equation: $\Delta \delta =\Delta {\delta }_{\max }\left(\frac{\left[\mathrm{L}\right]+\left[\mathrm{P}\right]+K_{\mathrm{d}}-{\left(\right[\mathrm{L}]+[\mathrm{P}]+K_{\mathrm{d}})}^2-4\left[\mathrm{P}\right]\left[\mathrm{L}\right]}{2\left[\mathrm{P}\right]}\right)$ where [L] is the concentration of ubiquitin, [P] is the concentration of REV1, Δδ is the observed normalized chemical shift change as defined in Fig. 1b, and Δδ_max is the normalized chemical shift change at saturation. (b) ITC of REV1 with ubiquitin. The integrated heat measurements from raw titration data and curve fitting using a one-site binding model are shown. The K_d and stoichiometry (n) values are indicated with the standard deviations determined by nonlinear least-squares fitting. (c) Representative intermolecular NOE signals identified in F1 ¹³C-filtered, F2 ¹³C-edited NOESY-HSQC spectra. The resonance labels are colored orange for REV1 and blue for ubiquitin.