Expression of isotope‐labeled ubiquitin for NMR

For the expression of ¹⁵N‐labeled ubiquitin monomers with and without 6xHis‐tag, cells were grown in minimal medium containing 7.5 g/l Na₂HPO₄, 3 g/l KH₂PO₄, 0.5 g/l NaCl, 2 mM MgSO₄, 0.1 mM CaCl₂, 10 µM FeSO₄, 4 g/l glucose, 1 g/l ¹⁵NH₄Cl, vitamin mix, and trace element mix. Vitamin mix final concentrations were 1 mg/l D‐biotin, 500 µg/l choline chloride, 500 µg/l folic acid, 1 mg/l myoinositol, 500 µg/l nicotinamide, 500 µg/l pantothenic acid, 500 µg/l pyridoxal hydrochloride, 50 µg/l riboflavin, and 500 µg/l thiamine hydrochloride. Trace element mix final concentrations were 50 mg/l EDTA, 8.3 mg/l FeCl₃ × 6H₂O, 840 µg/l ZnCl₂, 0.13 mg/l CuCl₂×2H₂O, 100 µg/l CoCl₂ × 6H₂O, 100 µg/l H₃BO₃, and 16 µg/l MnCl₂ × 6 H₂O.