Ascend 700 spectrometer

The optical rotations were measured in MeOH using a Rudolph Research Autopol III (Hackettstown, NJ, USA). UV spectra were recorded on a Hitachi JP/U-3010 UV spectrophotometer (Tokyo, Japan). All NMR spectra were recorded on a Bruker Ascend 700 spectrometer (Billerica Middlesex County, MA, USA) using DMSO-d₆ as solvent. Chemical shifts were reported with reference to the respective solvent peaks (δ_H 2.50 and δ_C 39.52 for DMSO-d₆ methanol-d₄). Electrospray ionization (ESI) source low-resolution mass data were obtained on an Agilent Technologies 6120 quadrupole mass spectrometer (Santa Clara, CA, USA) coupled with an Agilent Technologies 1260 series HPLC. High-resolution mass spectrometric data were collected on a JEOL JMS-700 double-focusing (B/E configuration) instrument (Tokyo, Japan). High-performance liquid chromatography (HPLC) was performed using a Waters HPLC (Milford Worcester County, MA, USA) equipped with a Waters 2998 photodiode array detector and Waters 1525 binary pump. HPLC-grade solvents (Daejeon, Korea) were used for HPLC. NMR solvents were purchased from Cambridge Isotope Laboratories Inc. (Andover, MA, USA).

Optical rotations were determined using a Perkin-Elmer (Überlingen, Germany) 241 spectrophotometer; ultraviolet (UV) spectra were recorded with a Shimadzu (Duisburg, Germany) UV-2450 UV-Vis spectrophotometer. NMR spectra were recorded on a Bruker (Bremen, Germany) Ascend 700 spectrometer equipped with 5 mm TXI cryoprobe (¹H-700 MHz, ¹³C-175 MHz) spectrometer. HR-ESI-MS mass spectra were recorded using a Bruker (Bremen, Germany) Agilent 1260 series HPLC-UV/Vis system (column 2.1 × 50 mm, 1.7 m, C18 Acquity UPLC BEH (waters), solvent A: H₂O + 0.1% formic acid; solvent B: AcCN + 0.1% formic acid, gradient: 5% B for 0.5 min increasing to 100% B in 19.5 min and then maintaining 100% B for 5 min, flow rate 0.6 mL/min, UV/Vis detection at 200–600 nm combined with ESI-TOF-MS (MaXiS, Bruker, Bremen, Germany) [scan range 100–2500 m/z, capillary voltage 4500 V, dry temperature 200 °C]. Chemicals and solvents were obtained from AppliChem GmbH (Darmstadt, Germany), Avantor Performance Materials (Arnhem, Netherlands), Carl Roth GmbH & Co. KG (Karlsruhe, Germany) and Merck KGaA (Darmstadt, Germany) in analytical and HPLC grade.

¹⁵N-labelled UBC9 proteins were prepared at 20 µM or 50 µM in a buffer containing 100 mM sodium phosphate, pH 6.0, 5 mM dithiothreitol, 0.02% NaN₃, and 10% D₂O^{42 (link)}. All NMR spectra were collected at 298 K on a Bruker Ascend 700 spectrometer. WT peptide (3.4 mM) and K689R peptide (2.4 mM) were stepwise titrated into UBC9 and chemical shift perturbation were monitored in series of the ¹H-¹⁵N HSQC spectra. The dissociation constant Kd was estimated using chemical shift changes as a function of the peptide:UBC9 molar ratios of the indicated residue in the proton dimension.

HRESIMS and HRESIMS/MS spectra were measured by a MaXis 4G UHR59 TOFMS spectrometer. All 1D and 2D NMR spectra were acquired by a Bruker Ascend 700 spectrometer (Bruker Company, Karlsruhe, Germany) with TMS as the internal standard (Sigma-Aldrich Inc., Vienna, Austria) Silica gel (100−200 mesh and 200−300 mesh; Yantai Jiangyou Silica Gel Development company, Yantai, China) was used for column chromatography. Analytic HPLC was performed by an Agilent 1260 HPLC system equipped with a G1311C isocratic pump and an Agilent G1315D diode array detector (DAD), using a reversed-phase column Basic C18 120A (Agilent company, 4.6 × 250 mm, 5 μm, Santa Clara, CA, USA). Semipreparative HPLC was performed by an Agilent 1260 HPLC system equipped with a 1110 isocratic pump and a 1430 DAD detector, using an ODS-A column (YMC company, 10 mm × 250 mm, 5 μm, Kyoto, Japan).