beta casein  (Thermo Fisher)

Journal: Protein Science : A Publication of the Protein Society

Article Title: Streptococcus pneumoniae HtrA is a dynamic and monomeric virulence factor capable of forming larger oligomeric complexes

doi: 10.1002/pro.70411

Figure Lengend Snippet: S. pneumoniae HtrA. AlphaFold predicted model of HtrA with key elements shown, including the N‐terminal PD and the C‐terminal PDZ. LoopA and the catalytic triad are also delineated. Like most HtrAs, S. pneumoniae HtrA is initially tethered to the membrane through an N‐terminal transmembrane region.

Article Snippet: Both the apo and β‐casein‐bound HtrA samples were imaged on a Titan Krios microscope at the Pacific Northwest Center for Cryo‐EM.

Techniques: Membrane

Journal: Protein Science : A Publication of the Protein Society

Article Title: Streptococcus pneumoniae HtrA is a dynamic and monomeric virulence factor capable of forming larger oligomeric complexes

doi: 10.1002/pro.70411

Figure Lengend Snippet: S. pneumoniae HtrA cleavage of caseins and preferred cleavage sites. (a) HtrA cleavage of bovine α‐casein (bov α), bovine β‐casein (bov β), and human β‐casein (hum β). (b) SDS–PAGE analysis of HtrA‐mediated β‐casein cleavage using the full HtrA ectodomain and independently produced PD and PDZ. (c) Counts derived from MS‐derived identification of HtrA cleavage peptides for P1 sites. (d) Counts derived from MS‐derived identification of HtrA cleavage peptides for P1′ sites. Concentrations of both HtrA and caseins were 10 μM with incubations at 37°C. (e) Schematic of general protease nomenclature illustrating the preferred preference for P1 and P1′ cleavage sites of S. pneumoniae HtrA identified here.

Article Snippet: Both the apo and β‐casein‐bound HtrA samples were imaged on a Titan Krios microscope at the Pacific Northwest Center for Cryo‐EM.

Techniques: SDS Page, Produced, Derivative Assay

Journal: Protein Science : A Publication of the Protein Society

Article Title: Streptococcus pneumoniae HtrA is a dynamic and monomeric virulence factor capable of forming larger oligomeric complexes

doi: 10.1002/pro.70411

Figure Lengend Snippet: Structural studies of S. pneumoniae HtrA constructs. Analytical size exclusion chromatography (Superdex‐75) and 15 N‐HSQC spectra are shown for (a) Analytical sizing (Superdex‐75) and 15 N‐HSQC spectra (900 MHz) are shown for the full HtrA ectodomain of residues 59–393. This construct eluted at 10.4 mL that corresponds to 44 kDa estimated from a standard curve, while its real molecular weight is 36 kDa. (b) Analytical sizing (Superdex‐75) and 15 N‐HSQC spectra (900 MHz) are shown for the HtrA PD, residues 59–285. This construct eluted at 11.8 mL that corresponds to 24 kDa estimated from a standard curve, while its real molecular weight is 23 kDa. (c) Analytical sizing (Superdex‐75) and 15 N‐HSQC spectra (900 MHz) are shown for the HtrA PDZ, residues 285–393. This construct eluted at 13.7 mL that corresponds to 11 kDa estimated from a standard curve, while its real molecular weight is 12 kDa. (d) CA chemical shift differences to that of a random coil (Δ δ Cα) for the HtrA PD. (e) CA chemical shift differences to that of a random coil (Δ δ Cα) for the HtrA PDZ. (f) Secondary structure propensities were calculated from all backbone assignments using the Chemical Shift Index (CSI) and are delineated as β‐strand (green) or α‐helix (red). From CSI, β‐strand includes the following: 73–78, 102–110, 114–120, 130–134, 139–146, 153–158, 164–168, 180–185, 197–201, 207–210, 218–225, 238–241, 244–250, 265–269, 294–297, 318–323, 339–343, 366–373, 376–387. From CSI, α‐helix includes the following: 60–67, 270–283, 303–308, and 351–359.

Article Snippet: Both the apo and β‐casein‐bound HtrA samples were imaged on a Titan Krios microscope at the Pacific Northwest Center for Cryo‐EM.

Techniques: Construct, Size-exclusion Chromatography, Molecular Weight

Journal: Protein Science : A Publication of the Protein Society

Article Title: Streptococcus pneumoniae HtrA is a dynamic and monomeric virulence factor capable of forming larger oligomeric complexes

doi: 10.1002/pro.70411

Figure Lengend Snippet: NMR titrations of the independently folded S. pneumoniae HtrA subdomains. (a) Selected regions illustrating titrations of 15 N‐labeled PD titrated with unlabeled PDZ (left) and the reverse titration (right). Schematics illustrating the titration is also included. Spectra include the following: Free (black), 0.25 mM (blue, only in PD titration with PDZ), 0.5 mM (sky blue), 1 mM (green), 2 mM (mauve), and 4 mM (red). (b) Binding isotherms for each titration were used to quantify the binding affinities from multiple resonance CSPs. From global fits for each titration, a K D of 480 ± 43 μM was derived (left) and a 1.0 ± 0.5 mM was derived (right). (c) CSPs plotted for each titration between no titrant and 4 mM titrant for the PD (left) and the PDZ (right). For the PD domain (left), the average CSP was 0.006 ppm and standard deviation 0.013 ppm. This average plus a half standard deviation of 0.013 ppm is delineated (dashed line). For the PDZ domain (right), the average CSP was 0.44 ppm and standard deviation 0.41 ppm. The average plus a half standard deviation is of 0.63 ppm is delineated (dashed line). (d) From titrations of the independent domains, CSPs larger than the average plus one standard deviation are mapped onto the HtrA model (blue spheres). All NMR data were collected at 900 MHz at 35°C using 500 μM labeled domains.

Article Snippet: Both the apo and β‐casein‐bound HtrA samples were imaged on a Titan Krios microscope at the Pacific Northwest Center for Cryo‐EM.

Techniques: Labeling, Titration, Binding Assay, Derivative Assay, Standard Deviation

Journal: Protein Science : A Publication of the Protein Society

Article Title: Streptococcus pneumoniae HtrA is a dynamic and monomeric virulence factor capable of forming larger oligomeric complexes

doi: 10.1002/pro.70411

Figure Lengend Snippet: Spectral comparisons and relaxation experiments of S. pneumoniae HtrA. (a) Examples of systematic shifts for LoopA residues within the free PD (black), the full ectodomain (green), and the biochemically saturated PD with 4 mM PDZ (red) for F87, G88, and N89. A schematic illustrating the potential model of HtrA existing as “opened” and “closed” is illustrated based on the observation that resonances of the full ectodomain fall in between. (b) R 2 ‐CPMG dispersions of the PD alone, the full ectodomain, and the biochemically saturated PD with a cartoon drawing above describing the observed exchange. (c) R 2 ‐CPMG dispersions of the full ectodomain. Shown are global fits for S74 (blue), V86 (blue), G126 (violet), D152 (cyan), G215 (maroon), T227 (red), I311 (brown), N360 (magenta), I369 (orange), and N379 (green). Further R 2 ‐dispersions are also provided (Figure ). (d) Resonances are mapped onto the predicted HtrA structure that exhibit R 2 ‐CPMG dispersion (green spheres), noting that backbone resonances still await completion for the full HtrA ectodomain. All data were collected at 900 MHz at 35°C.

Article Snippet: Both the apo and β‐casein‐bound HtrA samples were imaged on a Titan Krios microscope at the Pacific Northwest Center for Cryo‐EM.

Techniques: Dispersion

Journal: Protein Science : A Publication of the Protein Society

Article Title: Streptococcus pneumoniae HtrA is a dynamic and monomeric virulence factor capable of forming larger oligomeric complexes

doi: 10.1002/pro.70411

Figure Lengend Snippet: Global effects induced by the S. pneumoniae HtrA S234A mutant monitored via NMR. (a) Selected region of 15 N‐HSQC for PD WT (black) and PD S234A (purple), illustrating both shifts and the disappearance of resonances. (b) Global amide CSPs between PD WT and PD S234A (black) with resonances that either disappear or exhibit CSPs too large to identify (purple). (c) Examples of full HtrA S234A ectodomain induced CSPs (purple) relative to the full HtrA WT ectodomain (gray) that are in the same direction of the titrations of the PD (black) with 0.5 mM PDZ (blue), 1 mM PDZ (green), and 4 mM PDZ (red). (d) Examples of full HtrA S234A ectodomain induced CSPs (purple) relative to the full HtrA WT ectodomain (gray) that are in the opposite direction of the titrations of the PDZ (black) with 0.5 mM PD (blue), 1 mM PD (green), and 4 mM PD (red). (e) Amide CSPs between full HtrA WT ectodomain and the S234A mutation indicating measured CSPs (black spheres) and those that shift to unidentified positions (purple spheres) mapped onto the PD. Additional resonances within the PDZ domain in the context of the full HtrA ectodomain that shift with the S234A mutation are also shown (black spheres). The average CSP was 0.15 ppm and standard deviation was 0.14 ppm (dashed line indicates the sum).

Article Snippet: Both the apo and β‐casein‐bound HtrA samples were imaged on a Titan Krios microscope at the Pacific Northwest Center for Cryo‐EM.

Techniques: Mutagenesis, Standard Deviation

Journal: Protein Science : A Publication of the Protein Society

Article Title: Streptococcus pneumoniae HtrA is a dynamic and monomeric virulence factor capable of forming larger oligomeric complexes

doi: 10.1002/pro.70411

Figure Lengend Snippet: Cryo‐EM analysis of the S. pneumoniae HtrA S234A mutant‐induced oligomer. (a) Size‐exclusion chromatography of HtrA WT, HtrA S234A, β‐casein, and a mixture of HtrA S234A with excess β‐casein. (b) Cryo‐EM density map and corresponding atomic model of substrate‐free HtrA S234A with the side view of trimer/trimer interaction. (c) Cryo‐EM density map and corresponding atomic model of the HtrA S234A/β‐casein complex with the side view of trimer/trimer interaction. (d) Local resolution of apo S. pneumoniae HtrA S234A colored at 3–4 Å (red), 4–5 Å (yellow), and 5–6.5 Å (green). (e) Local resolution of the HtrA S234A/β‐casein complex colored at 3–4 Å (red), 4–5 Å (yellow), and 5–6.5 Å (green). (f) Model of the apo S. pneumoniae HtrA S234A trimer in the same orientation as (d). (g) Model of the HtrA S234A/β‐casein complex trimer in the same orientation as (e).

Article Snippet: Both the apo and β‐casein‐bound HtrA samples were imaged on a Titan Krios microscope at the Pacific Northwest Center for Cryo‐EM.

Techniques: Cryo-EM Sample Prep, Mutagenesis, Size-exclusion Chromatography

Journal: Protein Science : A Publication of the Protein Society

Article Title: Streptococcus pneumoniae HtrA is a dynamic and monomeric virulence factor capable of forming larger oligomeric complexes

doi: 10.1002/pro.70411

Figure Lengend Snippet: S. pneumoniae HtrA domain‐specific interactions with β‐casein. (a) Selected region of 15 N‐HSQC for 250 μM HtrA PD alone (black) and in the presence of 500 μM full‐length β‐casein (red). (b) Selected region of 15 N‐HSQC for 250 μM HtrA PDZ alone (black) and in the presence of 500 μ full‐length β‐casein (red). (c) Selected region of 15 N‐HSQC for a titration of 250 μM HtrA PDZ with recombinantly purified regions of β‐casein that include residues 21–70 (left), residues 71–130 (middle), and residues 101–224 (right). Intermediate titration points are only shown for β‐casein residues 21–70, while only the free and final 400 mM concentrations of added β‐casein fragments are shown for the others. (d) Binding isotherms of 15 N‐PDZ from titrations with β‐casein residues 21–70 are shown that were globally fit with an extracted K D of 215 ± 35 μM. Only a subset of the nearly two dozen amide shifts are shown for clarity. (e) Amide CSPs between 15 N‐PDZ alone and in the context of 400 μM β‐casein residues 21–70. The average CSP was 0.10 ppm with a standard deviation of 0.10 ppm. Dashed lines correspond to the average plus one standard deviation (0.20 ppm, green) and ½ standard deviation (0.15 ppm, blue). (f) CSPs are mapped onto the HtrA PDZ within the model structure with spheres color‐coded and described in (e). (g) SDS–PAGE analysis of full HtrA ectodomain and each of the three β‐casein constructs are shown both alone and incubated together. β‐Casein residues 21–70 and residues 71–130 also comprise the hSUMO tag for visualization on the gel and β‐casein residues 101–224 comprises an N‐terminal 6xHis tag.

Article Snippet: Both the apo and β‐casein‐bound HtrA samples were imaged on a Titan Krios microscope at the Pacific Northwest Center for Cryo‐EM.

Techniques: Titration, Purification, Binding Assay, Standard Deviation, SDS Page, Construct, Incubation

Journal: Protein Science : A Publication of the Protein Society

Article Title: Streptococcus pneumoniae HtrA is a dynamic and monomeric virulence factor capable of forming larger oligomeric complexes

doi: 10.1002/pro.70411

Figure Lengend Snippet: Proposed model of S. pneumoniae HtrA interactions. (a) S. pneumoniae HtrA dynamically samples both open and closed conformations in solution. The closed conformation was modeled using Swiss‐Model based on the structure of human HtrA2 (PDB ID 1ICY ). (b) The HtrA monomer initially engages substrates such as β‐casein through the substrate N‐terminal residues 21–70 (left), but can assemble into higher‐order oligomers, including hexameric complexes, in the presence of specific substrates (right). The hexameric complex represents that from the S. pneumoniae HtrA S234A mutant with β‐casein.

Article Snippet: Both the apo and β‐casein‐bound HtrA samples were imaged on a Titan Krios microscope at the Pacific Northwest Center for Cryo‐EM.

Techniques: Mutagenesis