Fig. 2: Using high-throughput proteomics data to detect native NCLE misfolding.
a Schematic overview of the limited proteolysis mass spectrometry experiments16. This method involves splitting a sample of the E. coli proteome in two, chemically denaturing one of the samples overnight followed by initiating refolding through a 100-fold dilution. The sample is then allowed to equilibrate for either 1 min, 5 min, or 2 h, followed by exposure to proteases that cleave surface-exposed residues. The resulting peptide fragments are then identified and their abundances measured via mass spectrometry. This approach detects structural changes within individual proteins at the residue level by comparing differences in the abundances of peptides containing proteinase-K cut-sites observed between the treated sample and the untreated sample. If the patterns are different then the locations of those differences reflect non-native structure at those residues, which we refer to as significant cut-sites. As in Fig. 1, red and blue regions indicate the loop (closed by the native contact shown as orange spheres) and threading regions of a native non-covalent lasso entanglement (NCLE). Dashed lines indicate a non-native NCLE change. White triangles indicate proteinase-K cut-sites that have no statistical difference in their abundance between the untreated and treated samples, while gray triangles exhibit a statistically significant change in abundance. Resulting half-tryptic peptides with significant changes in abundance after false discovery rate correction (black triangles) are mapped to the primary structure of E. coli proteins and, in this study, cross-referenced against various NCLE locations and features. b Summary of the native NCLE and essentiality status of the mass-spec observed proteins in the absence of chaperones (cyto-serum) and the presence of DnaK and GroEL. c Possible NCLE misfolding mechanisms of a protein containing a native NCLE, and a protein absent a native NCLE.
