malt protein  (GE Healthcare)

Journal: Nature Communications

Article Title: Structural basis for negative regulation of the Escherichia coli maltose system

doi: 10.1038/s41467-023-40447-y

Figure Lengend Snippet: a Affinity-purified MalT and MalY proteins were further analyzed by gel filtration using a Superdex 200 Increase 10/300 GL column in the presence of ADP (left). When MalT and MalY were incubated in a 1:1 molar ratio, the two proteins formed a stable complex with a molecular weight of about 300 kDa. The 9-mL peak observed when MalT is filtered alone may contain protein aggregates or MalT protein that partially oligomerized at a high protein concentration (>3 mg/ml). The presence of MalT and MalY in fractionated samples was confirmed by SDS-PAGE analyses (right). The experiments have been repeated for three times with similar results. b Cryo-EM density map with 2.94 Å resolution (top) and the refined structure model (middle) of the MalT-MalY complex (PDB: 8BOB) shown in different orientations. Each MalT or MalY protomer is labelled. Residue number and colors of each protein domain are indicated (bottom). The N-terminal segment of NBD is highlighted in yellow. The C-terminal domains of MalT are not well defined in the final structure. DBD DNA-binding domain.

Article Snippet: To reconstitute the nucleoprotein complex, promoter DNA was mixed with purified WT or mutant MalT proteins at stoichiometric ratio (1:5) in 20 μl of 1× buffer I and incubated at 20 °C for 20 min. After addition of 5× native gel sample buffer (250 mM Tris-base, 250 mM boric acid, 5 mM EDTA, 50% Glycerol, 0.1% bromophenol blue), samples were loaded on a 6% native PAGE gel (Thermo Scientific) and electrophoresed at room temperature in 0.5× TBE buffer (50 mM Tris-base, 50 mM boric acid, 1 mM EDTA).

Techniques: Affinity Purification, Filtration, Incubation, Molecular Weight, Protein Concentration, SDS Page, Cryo-EM Sample Prep, Residue, Binding Assay

Journal: Nature Communications

Article Title: Structural basis for negative regulation of the Escherichia coli maltose system

doi: 10.1038/s41467-023-40447-y

Figure Lengend Snippet: a The interface between MalT and the PLP-binding domain of MalY with structural details. The MalT-recognizing surface patch on MalY is highlighted in red (right), polar interactions are represented by dashed lines, residues involved in hydrophobic packing are also shown (left). Each MalT or MalY protomer is labelled, and colors of each protein domain are indicated. The N-terminal segment of NBD is highlighted in yellow. b Pull-down assay using GST-tagged MalY protein and WT or mutant MalT proteins carrying interface mutations. The experiments have been repeated for three times with similar results. c Levels of β-galactosidase activity in strains G and H harboring WT MalT plasmid (pJB215) or a derivative thereof and grown in a minimal medium supplemented with glycerol. The enzymatic activity values obtained were corrected for the background as measured with strains harboring empty vector (pJM241). The values given are the means ± SD of results from three independent experiments. The asterisks indicate significance of two-tailed Student’s t -tests, *** P < 0.001. MalT proteins were detected by western blot using total-cell extracts from the assayed cultures. A nonspecific band with lower molecular weight that appeared in all the samples was used as loading control.

Article Snippet: To reconstitute the nucleoprotein complex, promoter DNA was mixed with purified WT or mutant MalT proteins at stoichiometric ratio (1:5) in 20 μl of 1× buffer I and incubated at 20 °C for 20 min. After addition of 5× native gel sample buffer (250 mM Tris-base, 250 mM boric acid, 5 mM EDTA, 50% Glycerol, 0.1% bromophenol blue), samples were loaded on a 6% native PAGE gel (Thermo Scientific) and electrophoresed at room temperature in 0.5× TBE buffer (50 mM Tris-base, 50 mM boric acid, 1 mM EDTA).

Techniques: Binding Assay, Pull Down Assay, Mutagenesis, Activity Assay, Plasmid Preparation, Two Tailed Test, Western Blot, Molecular Weight, Control

Journal: Nature Communications

Article Title: Structural basis for negative regulation of the Escherichia coli maltose system

doi: 10.1038/s41467-023-40447-y

Figure Lengend Snippet: The structure of MalT NBD-HD and one interacting MalY molecule (shown in transparent surface view) from the MalT-MalY complex was aligned to ( a ), lateral dimers comprising the NBD and HD of Apaf-1, ZAR1, and CED-4, or ( b ), an inactive NLRC4 consisting of its NBD, HD1, and LRR. c A same molar amount of MalT WT, R143A, R173A, and R143A/R173A proteins were pre-incubated and subjected to gel filtration analyses using a Superose 6 Increase 10/300 GL column in the presence of 1 mM maltotriose and 0.4 mM ATP. d A same molar amount of MalT and MalY WT or MalY-D182A/E185A/S218A proteins were preincubated and subjected to gel filtration analyses using a Superdex 200 Increase 10/300 GL column in the presence of 1 mM maltotriose and 0.1 mM ADP, either separately or together (left). The presence of MalT and MalY in fractionated samples was confirmed by SDS-PAGE analyses (right). The experiments have been repeated for three times with similar results.

Article Snippet: To reconstitute the nucleoprotein complex, promoter DNA was mixed with purified WT or mutant MalT proteins at stoichiometric ratio (1:5) in 20 μl of 1× buffer I and incubated at 20 °C for 20 min. After addition of 5× native gel sample buffer (250 mM Tris-base, 250 mM boric acid, 5 mM EDTA, 50% Glycerol, 0.1% bromophenol blue), samples were loaded on a 6% native PAGE gel (Thermo Scientific) and electrophoresed at room temperature in 0.5× TBE buffer (50 mM Tris-base, 50 mM boric acid, 1 mM EDTA).

Techniques: Incubation, Filtration, SDS Page

Journal: Nature Communications

Article Title: Structural basis for negative regulation of the Escherichia coli maltose system

doi: 10.1038/s41467-023-40447-y

Figure Lengend Snippet: a The first 20 amino acids N-terminal to the NBD of MalT form a loop structure (highlighted in yellow) and make extensive contacts with both NBD and WHD. Polar interactions are represented by dashed lines, colors of each protein domain are indicated. b In vivo activities of MalT WT and N-loop mutants. The levels of β-galactosidase activities were determined by using strain H harboring pJB215 or a derivative thereof and corrected for the background as described in Fig. . The values given are the ratios of the mutant activities to that of WT. The relative protein levels of MalT mutant to that of WT were determined by western blot quantification using total-cell extracts from the assayed cultures. A non-specific band with lower molecular weight that appeared in all the samples was used as loading control. All the values are the means ± SD of results from three independent experiments. c Luciferase assays of MalT WT and N-loop mutants. Proteins were first purified in the presence of 0.4 mM ATP. Free nucleotides were then removed from the samples by gel filtration, the protein-bound nucleotides were released, and their amounts quantified. The nucleotide content was calculated as molar percentage of nucleotide to protein. All the values are the means ± SD of results from three independent experiments.

Article Snippet: To reconstitute the nucleoprotein complex, promoter DNA was mixed with purified WT or mutant MalT proteins at stoichiometric ratio (1:5) in 20 μl of 1× buffer I and incubated at 20 °C for 20 min. After addition of 5× native gel sample buffer (250 mM Tris-base, 250 mM boric acid, 5 mM EDTA, 50% Glycerol, 0.1% bromophenol blue), samples were loaded on a 6% native PAGE gel (Thermo Scientific) and electrophoresed at room temperature in 0.5× TBE buffer (50 mM Tris-base, 50 mM boric acid, 1 mM EDTA).

Techniques: In Vivo, Mutagenesis, Western Blot, Molecular Weight, Control, Luciferase, Purification, Filtration

Journal: Nature Communications

Article Title: Structural basis for negative regulation of the Escherichia coli maltose system

doi: 10.1038/s41467-023-40447-y

Figure Lengend Snippet: a In vivo activities of WT MalT or N-loop mutants that abolished protein oligomerization. β-galactosidase activities were determined by using strain H harboring pJB215 or a derivative thereof and corrected for the background as described in Fig. . The values given are the ratios of the mutant activities to that of WT. The relative protein levels of MalT mutant to that of WT were determined by western blot quantification using total-cell extracts from the assayed cultures. A non-specific band with lower molecular weight that appeared in all the samples was used as loading control. All the values are the means ± SD of results from three independent experiments. b A same molar amount of MalT WT or N-loop mutant proteins were pre-incubated and subjected to gel filtration analyses using a Superose 6 Increase 10/300 GL column in the presence of 1 mM maltotriose and 0.4 mM ATP. c Luciferase assays of WT MalT or N-loop mutants that abolished protein oligomerization. The assays were done as described in Fig. . All the values are the means ± SD of results from three independent experiments. d A modeled MalT dimer containing NBD and HD. The N-loop is highlighted in yellow. The structure was modeled based on an Apaf-1 lateral dimer from the Apaf-1 apoptosome. Both the N-terminal loop and MalY-interacting interface of MalT are in the dimerization surface in the modeled structure. Residues contributing to protein oligomerization are shown.

Article Snippet: To reconstitute the nucleoprotein complex, promoter DNA was mixed with purified WT or mutant MalT proteins at stoichiometric ratio (1:5) in 20 μl of 1× buffer I and incubated at 20 °C for 20 min. After addition of 5× native gel sample buffer (250 mM Tris-base, 250 mM boric acid, 5 mM EDTA, 50% Glycerol, 0.1% bromophenol blue), samples were loaded on a 6% native PAGE gel (Thermo Scientific) and electrophoresed at room temperature in 0.5× TBE buffer (50 mM Tris-base, 50 mM boric acid, 1 mM EDTA).

Techniques: In Vivo, Mutagenesis, Western Blot, Molecular Weight, Control, Incubation, Filtration, Luciferase

Journal: Nature Communications

Article Title: Structural basis for negative regulation of the Escherichia coli maltose system

doi: 10.1038/s41467-023-40447-y

Figure Lengend Snippet: a 2D classification from cryo-EM analyses of active MalT. The density of C-terminal domains is indicated by red empty arrows. Particles show a preferred orientation. b A model for MalT regulation. In the absence of substrate transport, the idling E. coli maltose transporter MalFGK 2 anchors MalT to the cytoplasmic membrane via MalK-mediated interactions. Meanwhile, MalT molecules that remain in the cytoplasm are sequestered by inhibitory proteins. Sugar transport triggers a conformational change in the MalK dimer which frees membrane-localized MalT into cytoplasm , . The inhibitory effect of MalY can be relieved by an increased concentration of maltotriose. Binding of inducer to the sensor domain of MalT (1) is followed by a high-affinity binding step involving both the sensor and arm domains , driving the MalT activation pathway towards dissociation of the MalT-MalY complex (2), opening of NOD (3) and oligomerization. Maltotriose binding and oligomerization together stabilize the C-terminal domains of MalT, allowing the activator binding to promoter DNA and RNA polymerase (RNAP) recruitment for subsequent transcription initiation. The NBD, HD, WHD, arm, sensor, and DNA-binding domains of MalT are colored in pink, purple, wheat, cyan, slate, and green, respectively. Domains are colored in gray if they are flexible. This figure was created with BioRender.

Article Snippet: To reconstitute the nucleoprotein complex, promoter DNA was mixed with purified WT or mutant MalT proteins at stoichiometric ratio (1:5) in 20 μl of 1× buffer I and incubated at 20 °C for 20 min. After addition of 5× native gel sample buffer (250 mM Tris-base, 250 mM boric acid, 5 mM EDTA, 50% Glycerol, 0.1% bromophenol blue), samples were loaded on a 6% native PAGE gel (Thermo Scientific) and electrophoresed at room temperature in 0.5× TBE buffer (50 mM Tris-base, 50 mM boric acid, 1 mM EDTA).

Techniques: Cryo-EM Sample Prep, Membrane, Concentration Assay, Binding Assay, Activation Assay

Journal: Nature Communications

Article Title: Structural basis for negative regulation of the Escherichia coli maltose system

doi: 10.1038/s41467-023-40447-y

Figure Lengend Snippet: a Affinity-purified MalT and MalY proteins were further analyzed by gel filtration using a Superdex 200 Increase 10/300 GL column in the presence of ADP (left). When MalT and MalY were incubated in a 1:1 molar ratio, the two proteins formed a stable complex with a molecular weight of about 300 kDa. The 9-mL peak observed when MalT is filtered alone may contain protein aggregates or MalT protein that partially oligomerized at a high protein concentration (>3 mg/ml). The presence of MalT and MalY in fractionated samples was confirmed by SDS-PAGE analyses (right). The experiments have been repeated for three times with similar results. b Cryo-EM density map with 2.94 Å resolution (top) and the refined structure model (middle) of the MalT-MalY complex (PDB: 8BOB) shown in different orientations. Each MalT or MalY protomer is labelled. Residue number and colors of each protein domain are indicated (bottom). The N-terminal segment of NBD is highlighted in yellow. The C-terminal domains of MalT are not well defined in the final structure. DBD DNA-binding domain.

Article Snippet: Cryo-EM sample was prepared with purified MalT-MalY protein complex at a concentration of about 0.5 mg/ml and with MalT oligomer at 1 mg/ml.

Techniques: Affinity Purification, Filtration, Incubation, Molecular Weight, Protein Concentration, SDS Page, Cryo-EM Sample Prep, Binding Assay

Journal: Nature Communications

Article Title: Structural basis for negative regulation of the Escherichia coli maltose system

doi: 10.1038/s41467-023-40447-y

Figure Lengend Snippet: a 2D classification from cryo-EM analyses of active MalT. The density of C-terminal domains is indicated by red empty arrows. Particles show a preferred orientation. b A model for MalT regulation. In the absence of substrate transport, the idling E. coli maltose transporter MalFGK 2 anchors MalT to the cytoplasmic membrane via MalK-mediated interactions. Meanwhile, MalT molecules that remain in the cytoplasm are sequestered by inhibitory proteins. Sugar transport triggers a conformational change in the MalK dimer which frees membrane-localized MalT into cytoplasm , . The inhibitory effect of MalY can be relieved by an increased concentration of maltotriose. Binding of inducer to the sensor domain of MalT (1) is followed by a high-affinity binding step involving both the sensor and arm domains , driving the MalT activation pathway towards dissociation of the MalT-MalY complex (2), opening of NOD (3) and oligomerization. Maltotriose binding and oligomerization together stabilize the C-terminal domains of MalT, allowing the activator binding to promoter DNA and RNA polymerase (RNAP) recruitment for subsequent transcription initiation. The NBD, HD, WHD, arm, sensor, and DNA-binding domains of MalT are colored in pink, purple, wheat, cyan, slate, and green, respectively. Domains are colored in gray if they are flexible. This figure was created with BioRender.

Article Snippet: Cryo-EM sample was prepared with purified MalT-MalY protein complex at a concentration of about 0.5 mg/ml and with MalT oligomer at 1 mg/ml.

Techniques: Cryo-EM Sample Prep, Concentration Assay, Binding Assay, Activation Assay