FIGURE 9.

Assignment of HSQC spectrum of [¹⁵N]Ube2g1^C90S-UB^K48R oxyester. A, two sets of ¹H,¹⁵N HSQC peaks were identified resulting from the [¹⁵N]Ube2g1-UB^K48R oxyester and free [¹⁵N]Ube2g1 (labeled with f-), respectively. Nevertheless, the well dispersed spectrum of the purified [¹⁵N]Ube2g1^C90S-UB^K48R oxyester shows the absence of an intermolecular interaction between the attached donor ubiquitin and different Ube2g1 molecules. Inset, interestingly, the peak from the Gly-103 residue of the [¹⁵N]Ube2g1-UB^K48R oxyester was not strongly perturbed by additional free ubiquitin (0.6 mm) compared with that of free [¹⁵N]Ube2g1, suggesting that the Gly-103 residues were affected differently by added (i.e. acceptor) ubiquitin in the presence of the oxyester-attached donor ubiquitin. B, the HSQC-peaks assignment of [¹⁵N]Ube2g1^C90S-UB^K48R oxyester was performed by comparing the HSQC spectra with [¹⁵N]Ube2g1^C90S-UB^K48R oxyester and with [¹⁵N]Ube2g1^C90S hydrolyzed from its oxyester adduct. The residues displaying very similar peak positions in the HSQC spectrum for the oxyester and free form were assigned unambiguously, and some of additional residues were also assigned based on the HNCO spectrum. C, the CSPs of [¹⁵N]Ube2g1^C90S between the oxyester and free form are shown as a bar plot. The residues for which chemical shifts were too different between the oxyester and free form to be clearly assigned are indicated using green bars.