Figure 1.
Oligomeric state of DegPS210A in the presence of substrates of different sizes.
A. Elution profiles from a Superdex-200 column of solutions containing either DegPS210A alone (42 µM) (black line) or DegPS210A (42 µM) mixed with β-casein (42 µM) (blue line) or β-casein peptide fragments (168 µM) (red line). The expected elution volumes (Ve) for DegP6 (11.5 ml), DegP12 (9.9 ml) and DegP24 (8.4 ml) are indicated by the dashed lines in the plot. Numbers above the peaks refer to the gel lanes in the SDS-PAGE gel in panel B. B. SDS-PAGE analysis of fractions from the elution profiles in panel A. Lane 1, 2 and 3 resolved samples from fractions indicated with the corresponding numbers in the elution profiles in panel A. Similarly, lane 4 & 5 also contained samples from fractions indicated with the corresponding number in the elution profiles in panel A. However, these fractions were previously concentrated by Trichloroacetic acid precipitation (TCA ppt) to visualize the β-casein peptides. Lane 6 shows a sample of β-casein peptides used to form complexes. C. The DegPS210A:substrate complexes purified by size exclusion chromatography in (A) were visualized by negative staining EM. The left panels shows representative negatively stained electron micrographs obtained from the central fraction of the peaks labeled as “1&4” (top), “2” (middle) and “5” in the elution profiles in (A). The right panels shows a comparison of two-dimensional projections (column labeled as “Projection”) calculated from the X-ray structure of DegP24 (PDB ID: 3CS0) (middle panel) or the cryo-EM structure of DegP12 (PDB ID: 2ZLE) (bottom panel) with class averages (column labeled as “Average”) of the corresponding views calculated from particle images extracted from the micrographs in the left panel.
Figure 2.
Filtered β-casein peptide fragments induce DegP12 cages.
A. A 15% SDS-PAGE resolving a preparation of full-length β-casein (lines labeled as “β-casein”) and β-casein peptide fragments (lines labeled as “β-casein peptides”) before (−) and after (+) they were filtered through a 30 kDa-cutoff filter to remove full-length β-casein. Lines containing full-length β-casein were stained with Coomassie brilliant blue and lines containing the β-casein peptide fragments were silver stained to visualize that no full-length β-casein or proteolytically active DegP remained after filtration. B. Elution profiles from a Superdex-200 column of solutions containing DegPS210A (42 µM) mixed with a preparation of β-casein peptide fragments (168 µM) that has been filtered through a 30 kDa-cutoff filter to remove traces of full-length β-casein. The expected elution volumes (Ve) for DegP6 (11.5 mL), DegP12 (9.9 mL) and DegP24 (8.4 mL) are indicated by the dashed lines in the plot. C. Negatively stained electron micrograph obtained from the fraction at 10 mL from the size exclusion chromatography profile shown in panel B.
Figure 3.
Oligomeric state of DegPS210A in the presence of α-casein, malate dehydrogenase and their corresponding peptide fragments.
A. Coomassie brilliant blue stained 15% SDS-PAGE resolving the full-length α-casein and malate dehydrogenase (MDH) used to induce the formation of large oligomeric states in DegPS210A. Peptides generated from both proteins by incubation with DegP are also shown in the gel to provide an estimation of their molecular weight. B. Elution profiles from a Superdex-200 column of solutions containing either DegPS210A alone or DegPS210A mixed with α-casein or α-casein peptide fragments. The concentration of each component of the reaction is indicated. The expected elution volumes (Ve) for DegP6 (11.5 ml), DegP12 (9.9 ml) and DegP24 (8.4 ml) are indicated by the dashed lines in the plot. Numbers above the peaks refer to the electron micrographs in panel D. C. Elution profiles from a Superdex-200 column of solutions containing either DegPS210A alone or DegP210A mixed with MDH or MDH peptide fragments. The expected elution volumes (Ve) for DegP6 (11.5 ml), DegP12 (9.9 ml) and DegP24 (8.4 ml) are indicated by the dashed lines in the plot. Numbers above the peaks refer to the electron micrographs in panel D. D. Visualization of complexes of DegPS210A with α-casein and MDH substrates purified by size exclusion chromatography. The images are representative fields of negatively stained electron micrographs obtained from fractions labeled from 1 to 4 in the elution profiles shown in panels B and C. The arrowheads in panel 3 indicate some DegP12 cages present in this fraction.
Figure 4.
Oligomeric state of DegPS210A in the presence of PZ model peptides.
A. Names and sequences of the three PZ model peptides used to form complexes with DegPS210A in the size exclusion chromatography experiment shown in (B). Each peptide contains a protease motif at the N-terminus (labeled as “P”) and a PDZ motif at the C-terminus (labeled as “Z”). The +/− sign next to the “P” and “Z” labels indicated whether the motif is recognized (+) or not recognized (−) by the corresponding domain. B. Elution profiles from a Superdex-200 column of solutions containing DegPS210A alone (94 µM) or a mixture of DegPS210A (94 µM) with one of the model peptides at a concentration of 376 µM. The expected elution volumes (Ve) for DegP6 (11.5 ml) and DegP12 (9.9 ml) are indicated by the dashed lines in the plot.
Figure 5.
Oligomeric state of DegPS210A at various substrate concentrations.
Elution profiles of DegPS210A in the presence of various concentrations of β-casein peptides (A) or full-length β-casein (B). The expected elution volumes (Ve) for DegP6 (11.5 ml), DegP12 (9.9 ml) and DegP24 (8.4 ml) are indicated by the dashed lines in the plot. (C) Dynamic light scattering analysis of DegPS210A in the presence of variable concentrations of full-length β-casein or β-casein peptides. The graph shows the distribution of hydrodynamic radius of particles for various mixtures of DegPS210A and full-length β-casein or DegPS210A and β-casein peptides.
Figure 6.
Effect of temperature on the oligomeric state of DegPS210A:substrate complexes.
Representative electron micrographs of reaction mixtures containing DegPS210A alone (top panels), DegPS210A+β-casein (middle panels) or DegPS210A+β-casein peptides (bottom panels) at either 37°C (left panels) or 42°C (right panels). Complexes were assembled, applied to continuous carbon grids and negatively stained at the indicated temperatures.
Figure 7.
Cryo-electron microscopy of a reaction mixture containing DegPS210A and β-casein at high temperatures.
Representative micrographs of vitrified samples of a mixture of DegPS210A and β-casein at 37°C (A) and 42°C (B). Contrast of both images was inverted for easy comparison with negatively stained micrographs in previous figures. The right panels in (A) and (B) show a comparison of two-dimensional projections (column labeled as “Projection”) calculated from the X-ray structure of DegP24 (PDB ID: 3CS0) with class averages (column labeled as “Average”) of the corresponding views calculated from particle images extracted from the micrographs.