Protein Production.

The 11S RP from Trypanosoma brucei (perdeuterated) (44) was expressed with an N-terminal His₆-TEV sequence, T. acidophilum 20S CP β subunits (unlabeled) were expressed with an N-terminal NusA-His₆-TEV sequence, and T. acidophilum ILVM-¹³CH₃–labeled α-subunits included an N-terminal His₆-SUMO sequence. Finally, a His₆-SUMO-Strep-tag II-TEV sequence was added to the N terminus of α-subunits for expression in unlabeled media [Strep-tag II = Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (56)]. Protein expression was carried out using BL21 (DE3) Escherichia coli cells.

All unlabeled proteins were expressed in LB broth using an incubator shaker, expect for the β-subunits where expression was performed using TB broth via a single 10-L growth in a fermenter to maximize yield. Perdeuterated 11S and ILVM-¹³CH₃–labeled α-subunits were expressed in M9 minimal media, 99% D₂O, with d₇-glucose as the sole carbon source. For selective ILVM methyl labeling, 100 mg/L [ε-¹³C]-methionine (CLM-206-PK; Cambridge Isotope Laboratories), 60 mg/L α-ketobutyric acid (CDLM-7318; Cambridge Isotope Laboratories), and 100 mg/L α-ketoisovaleric acid (CDLM-7317; Cambridge Isotope Laboratories; one of the two isopropyl methyl groups is ¹³CH₃, the second is ¹²CD₃) were added 1 h before the induction of protein overexpression (52, 53). Cells were grown at 37 °C until OD ∼ 0.8, and then induced using 0.25 mM IPTG with overnight protein expression at room temperature. Cells resuspended in 50 mM sodium phosphate buffer, pH 7.8, 0.5 M NaCl, were lysed by sonication in the presence of DNase I and protease inhibitors, and the soluble cellular fraction was passed through a HisTrap column (GE Healthcare) and subsequently eluted with 0.3 M imidazole in 100 mM sodium phosphate buffer, pH 7.8. The N-terminal tags were then cleaved either with TEV protease (11S, β-subunit) or with UlP1 protease (α-subunit) and the protein passed again through the HisTrap column, collecting the flow-through. The final purification step involved size-exclusion chromatography with a Superdex 200 column (11S, α-subunit) in 50 mM sodium phosphate buffer, pH 7.8, 100 mM NaCl or anion exchange chromatography (β-subunit) using a HiTrap Q column (GE Healthcare) in Tris⋅HCl buffer, pH 8, and a NaCl gradient between 0 and 0.6 M.