Journal: Nature Communications

Article Title: Periplasmic protein quality control at atomic level in live cells

doi: 10.1038/s41467-025-62340-6

Figure Lengend Snippet: a Schematic representation of the protocol used for preparing bacterial samples overexpressing NDM-1 and the proteases Prc and DegP. Induction with arabinose (ARA) or IPTG was done at 37 ° C and 16 ° C, respectively. Incubation with spectinomycin (Spect.) was done at 20 ° C. b Comparison of NDM-1 overexpression levels from the in-cell NMR samples with P. aeruginosa and K. pneumoniae clinical strains ( N = 1). The dilution factor used (1/160) was determined by semi-quantitative western blot analysis and comparisons with the clinical strain Enterobacter cloacae 17464 (Supplementary Fig. ). Normalization was done considering the total number of cells estimated by OD 600 . c Membrane localization of overexpressed NDM-1 determined by Western blot analysis. Cyto., Perip., and Total memb. corresponds to cytosolic, periplasmic and total membrane fractions, respectively ( N = 3). d Representative immunofluorescence of NDM-1 (magenta) and Prc and DegP (green) in E. coli cells ( N = 3). e Mean fluorescence intensity of NDM-1 in E. coli cells. Panel shows a representative analysis of three independent repetitions ( N = 3). No NDM/DPA = 0.0011 ± 0.0001, NDM/No DPA = 2.8600 ± 0.2494, NDM/DPA = 1.6590 ± 0.1227 (mean ± SEM, n = 10 per treatment). Significant difference between conditions, **** p < 0.0001, Tukey, one-way ANOVA. f Western blot analysis of periplasmic NDM-1 degradation by Prc and DegP upon metal deprivation ( N = 3).

Article Snippet: Immunodetection of periplasmic maltose binding protein (MBP, Rockland Immunochemicals, 200-401-385) or cytoplasmic GroEL were performed as leakage or loading controls for periplasmic extracts or spheroplasts and whole cells, respectively.

Techniques: Incubation, Single Photon Emission Computed Tomography, Comparison, Over Expression, Western Blot, Membrane, Immunofluorescence, Fluorescence

Journal: Nature Communications

Article Title: Periplasmic protein quality control at atomic level in live cells

doi: 10.1038/s41467-025-62340-6

Figure Lengend Snippet: a 2D 1 H- 15 N SOFAST HMQC spectra of apoNDM-1 in E. coli cells when Prc and DegP were overexpressed (black) and at endogenous protease levels (light brown). Cyan circles identify NMR resonances with chemical shift displacements characteristic of newly generated C-terminal sites. Black arrows denote cross-peaks from amide groups of disordered peptides. The dotted square indicates tryptophan HN indole signals from newly generated apoNDM-1 peptide fragments. The vertical cyan line indicates the artifactual set of signals (T1 noise) caused by the sharp and intense resonance of unlabeled DPA. b Schematic representation of the resulting chemical groups upon amide peptide bond cleavage. c , d Representative time course of periplasmic apoNDM-1 degradation followed by 1D 1 H NMR analysis of tryptophan HN indole signals ( c ). Experiment was performed on two independent bacterial samples. Tryptophan HN indole NMR signal amplitude plateaus at c.a. 6.5 h ( d ). Each value corresponds to the normalized intensity at each timepoint. Error bars represent the experimental noise in each spectrum. e 13 C detected CBCACO (black) and CACO (cyan) NMR spectra of E. coli cells overexpressing NDM-1 and Prc/DegP after DPA treatment or in its absence (yellow). 13 CO NMR signals above 180 ppm belong to newly generated C-terminal backbone carboxylates. Cα and Cβ chemical shifts in the second spectral dimension identify the type of amino acid side chain. (*) indicate unassigned minor sites. In all cases, NMR spectra were registered at 20 °C.

Article Snippet: Immunodetection of periplasmic maltose binding protein (MBP, Rockland Immunochemicals, 200-401-385) or cytoplasmic GroEL were performed as leakage or loading controls for periplasmic extracts or spheroplasts and whole cells, respectively.

Techniques: Generated