Fig. 1: Design of GBC RNC constructs, solution-state NMR investigation of full-length GBC, and LC-MS/MS investigation of RNC fragments.
From: Cysteine oxidation and disulfide formation in the ribosomal exit tunnel
a Different lengths of GBC were fused at the N-terminal with a 10x histidine purification tag (white) and at the C-terminal with a 17-amino acid SecM sequence (dark gray). Numbering excludes the N-terminal methionine of GBC. GBC residues 1–32 (light blue), 33–43 (blue). 13C-labeled Cys residues are marked with an asterisk (*) and highlighted in yellow. Number of GBC RNCs residues are given from the Peptidyl transferase center (PTC) to the N-terminus excluding the 10x histidine purification tag (ΔPTC residues). b Lengths of the RNC constructs shown on the full-length GBC structure (PDB 4W9A) and 2D backbone 1H-15N-HSQC NMR spectrum of cysteine residues of full-length GBC reduced (red) and after oxidation with Cu(II) (black). Cysteine residues not changing position after oxidation (Cys18, Cys109) or that could not be unambiguously identified due to overlapping signals (Cys32) were labeled only once. The complete 1H-15N-HSQC NMR spectrum is shown in Supplementary Fig. 3. c Cα and Cβ chemical shifts of the six cysteines within full-length GBC reduced and after oxidation with Cu(II) compared to values derived from a chemical shift database24. Ellipses contain 90% of the corresponding chemical shifts of each group24 (H = helix, B = β-strand, C = coil). The spectral region for oxidized Cβ cysteines is highlighted in light gray, and for reduced cysteine in dark gray. The overlapping region of both states is shown in white. d Fragment spectrum of the tryptic peptide GFQGHAYECSSDAPNLQPYFSR (precursor m/z 695.54 (4+)) from U32SecM C18 with a glutathione modification on Cys18. e Fragment spectrum of the tryptic peptide GFQGHAYECSSDAPNLQPYFSR (precursor 835.03 (3+) m/z) from U32SecM C18 with a nitroso modification on Cys18. f Fragment spectrum of the tryptic peptide CNSIRVDSGCWMFSTPVWISQAQGIR (precursor m/z 980.46 (3+)) from U43SecM with a loop-linked disulfide bond (Cys32–Cys41). Matched fragment ions are indicated by dashes in the sequence and respective labels on peaks. g Overview of cysteine oxidation states (S-nitrosylation, S-glutathionylation, disulfide bonding) of the NCs of all RNCs (U32SecM, U43SecM, and U78SecM and mutants) as observed by LC/MS-MS (Supplementary Table 1). Trypsin cleavage site is shown as dashed line, disulfide bond as yellow line.
