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1.
Figure 2

Figure 2. Endogenously Generated ADP Stimulates PhoQ’s Phosphatase Activity In Vitro. From: Intrinsic Negative Feedback Governs Activation Surge in Two-Component Regulatory Systems.

(A) Levels of PhoP-P and PhoQ-P after incubation of PhoP with wild-type PhoQ protein in the presence of ATP (50 μM) for the indicated times.
(B) Quantitation of the phosphorylation assay shown in (A). The graph depicts the levels of PhoP-P (open circles) and PhoQ-P (closed circles) relative to their respective maximum.
(C) Levels of[α-32P] ATP and [α-32P] ADP after incubation of PhoP-P, PhoQ-P, and [α-32P] ATP for the indicated times. [α-32P] ATP was used as a standard marker for PEI-TLC.
(D) The graph depicts the levels of [α-32P] ATP and [α-32P] ADP from [α-32P] ATP during the reaction relative to the maximum levels shown in (C).
(E) Levels of PhoQ-P remain constant during autophosphorylation when PhoP is omitted from the reaction (A).
All data correspond to the average ± SEM from three independent experiments. See also Tables S3 and S4.

Won-Sik Yeo, et al. Mol Cell. ;45(3):409-421.
2.
Figure 1

Figure 1. Purified PhoQ and PhoP Proteins Recapitulate the PhoP-P Surge in the Presence of ATP and ADP In Vitro. From: Intrinsic Negative Feedback Governs Activation Surge in Two-Component Regulatory Systems.

(A) Levels of PhoP-P and PhoQ-P at the indicated times after incubation of PhoP with wild-type PhoQ protein in the presence of ATP (1 mM) and ADP (0.1 mM).
(B) Quantitation of the in vitro surge assay shown in (A). The graph depicts the absolute amount of PhoP-P (open circles) and PhoQ-P (closed circles). Inset: The normalized levels of PhoP-P (open circles) and PhoQ-P (closed circles) relative to their maximum levels. Data correspond to the average ± SEM from three independent experiments.
(C) ATP remaining in the reaction after incubation with PhoP and PhoQ proteins. ATP and Pi were visualized following a run on PEI-cellulose TLC plate. [γ-32P] ATP was used as a marker. The positions of ATP, Pi, PhoP-P, and PhoQ-P are indicated by arrows.
(D) Levels of PhoP and PhoQ proteins during in vitro surge were visualized on SDS-PAGE. The positions of PhoP and PhoQ are marked with arrows and a standard protein size marker was run in parallel.
See also Figure S1 and Tables S3 and S4.

Won-Sik Yeo, et al. Mol Cell. ;45(3):409-421.
3.
Figure 5

Figure 5. The ATP Lid S313A Mutant PmrB Displays Lower Binding Affinity for ADP and Decreased Phosphatase Activity. From: Intrinsic Negative Feedback Governs Activation Surge in Two-Component Regulatory Systems.

(A) [α-32P] ADP bound by wild-type (closed circles) and lid S313A mutant (open circles) PmrBc proteins at different ADP concentrations after UV crosslinking.
(B) Percentage of PmrA-P protein remaining after incubation with wild-type or S313A PmrBc proteins in the absence or presence of ADP (1 mM).
(C) The S313A PmrBc exhibits higher apparent KM for ADP than the wild-type PmrBc. Percentage of PmrA-P remaining after incubation with wild-type or S313A PmrBc proteins in the presence of the indicated ADP concentrations relative to the levels obtained in the absence of ADP.
(D) The S313A PmrBc dephosphorylates PmrA-P more slowly than the wild-type PmrBc. Percentage of PmrA-P remaining relative to the levels of PmrA-P at time zero after incubation with wild-type or S313A PmrBc proteins in the absence or presence of ADP (0.3 mM) for the indicated times.
(E) The wild-type and S313A PmrBc proteins display similar autokinase activity. Percentage of wild-type and S313A PmrBc after incubation in the presence of the indicated concentrations of ATP.
(F) The wild-type and S313A PmrBc proteins phosphorylate PmrA at similar rates. Percentage of PmrA-P after incubation with wild-type or S313A PmrBc proteins in the presence of ATP (1 mM).
All data correspond to the average ± SEM from three independent experiments. See also Tables S1, S3, and S4 and Figures S2–S4.

Won-Sik Yeo, et al. Mol Cell. ;45(3):409-421.
4.
Figure 4

Figure 4. Purified T438A Mutant PhoQ Displays Sustained PhoP-P Surge in the Presence of ATP and ADP In Vitro. From: Intrinsic Negative Feedback Governs Activation Surge in Two-Component Regulatory Systems.

(A) Levels of PhoP-P and PhoQ-P at the indicated times after incubation of PhoP with T438A mutant PhoQ protein in the presence of ATP (1 mM) and ADP (0.1 mM) (top). PhoP and PhoQ proteins during the in vitro surge were visualized on SDS-PAGE (bottom). The positions of PhoP and PhoQ were marked as arrows and a standard protein size marker was run in parallel.
(B) Quantitation of the in vitro surge assay shown in (A). The graph depicts the absolute amount of PhoP-P from PhoQ-T438A-P (open circles) and PhoQ-T438A-P (closed circles). Inset: the normalized levels of PhoP-P (open circles) and PhoQ-T438A-P (closed circles) relative to their maximum levels.
(C) Quantitative comparison of the in vitro surge assays shown in (Figures 1A and 4A). The graph depicts the absolute amounts of PhoP-P from either PhoQ-P (closed squares)orPhoQ-T438A-P (open squares) relative to their respective maximum levels. Inset: The normalized levels of PhoP-P from either PhoQ-P (closed squares) or PhoQ-T438A-P (open squares) relative to their maximum levels.
(D) Levels of PhoP-P from PhoQ-T438A-P and PhoQ-T438A-P after incubation of PhoP with T438A mutant PhoQ protein in the presence of ATP (50 μM) for the indicated times.
(E) Quantitation of the phosphorylation assay shown in (D). The graph depicts the levels of PhoQ-T438A-P (open circles) and PhoP-P from PhoQ-T438A-P (closed circles) relative to their respective maximum levels.
(F) Quantitative comparison of the phosphorylation assays shown in (Figures 2A and 4D). The graph depicts the levels of wild-type PhoQ-P (closed circles) and PhoQ-T438A-P (open circles) relative to their respective maximum levels.
(G) Quantitative comparison of the phosphorylation assay shown in (Figures 2A and 4D). The graph depicts the levels of PhoP-P from either PhoQ-P (closed squares) or PhoQ-T438A-P (open squares) relative to their respective maximum levels.
All data correspond to the average ± SEM from three independent experiments. See also Tables S3 and S4.

Won-Sik Yeo, et al. Mol Cell. ;45(3):409-421.
5.
Figure 7

Figure 7. A Salmonella Strain Expressing the T438A PhoQ Protein Exhibits Sustained Expression of PhoP-Activated Genes In Vivo. From: Intrinsic Negative Feedback Governs Activation Surge in Two-Component Regulatory Systems.

mRNA levels of the PhoP-activated pmrD, mgtA, pcgL, and mig-14 genes were determined by quantitative real-time PCR analysis with RNA prepared from strains WY105 (wild-type PhoQ)orWY107 (T438A mutant PhoQ) grown in medium with high (10 mM) Mg2+, shifted to medium containing low (33 μM) Mg2+ in the presence of 0.5 mM IPTG, and harvested at the designated times. mRNA levels were normalized to those of the 16S ribosomal RNA gene. Data are represented as ± SEM from three independent experiments. Kinetics based on the wild-type strain exhibit a surge where the mRNA levels increase, peak, and then decline within 45 min) (F statistics, p value < 0.03) in contrast to the behavior of the T438A-expressing strain. See also Tables S3 and S4.

Won-Sik Yeo, et al. Mol Cell. ;45(3):409-421.
6.
Figure 3

Figure 3. The ATP Lid T438A Mutant PhoQ Exhibits Lower Binding Affinity for ADP and Decreased Phosphatase Activity. From: Intrinsic Negative Feedback Governs Activation Surge in Two-Component Regulatory Systems.

(A and B) Binding affinity for ADP of the wild-type PhoQ protein (A) is higher than that of the lid T438A mutant PhoQ protein (B). The raw data (top) and corresponding integrated data (bottom) of heat changes are shown. In these ITC experiments, 2 mM and 5 mM of ADP were used for wild-type and T438A PhoQ proteins, respectively.
(C) ADP stimulates PhoQ’s phosphatase activity, and the T438A mutant is defective in phosphatase activity. Top: Levels of PhoP-P after incubation with wild-type or T438A PhoQ proteins in the absence or presence of ADP (1 mM). Bottom: Percentage of remaining PhoP-P relative to the levels present at time zero. PhoP-P levels at time zero min (control) are similar to those observed at time five min when no PhoQ protein was added to the reaction.
(D) The T438A PhoQ protein exhibits higher apparent KM for ADP in the in vitro phosphatase assay. Percentage of remaining PhoP-P after incubation with wild-type or T438A PhoQ proteins in the presence of the indicated ADP concentrations relative to the levels obtained in the absence of ADP.
(E) The T438A PhoQ protein dephosphorylates PhoP-P more slowly than the wild-type PhoQ protein. Percentage of PhoP-P remaining after incubation with wild-type or T438A PhoQ proteins in the absence or presence of ADP (1 mM) for different times relative to the levels of PhoP-P at time zero.
(F) The wild-type and T438A PhoQ proteins exhibit similar autokinase activity. Levels of phosphorylated wild-type and T438A PhoQ after incubation in the presence of the indicated ATP concentrations. The plot depicts the levels of PhoQ-P relative to the maximum.
(G) The wild-type and T438A PhoQ proteins autophosphorylate at similar rates. Percentage of phosphorylated wild-type and T438A PhoQ proteins relative to the maximum achieved after incubation in the presence of ATP (1 mM) for the indicated times.
(H) The wild-type and T438A PhoQ proteins phosphorylate Pho P at similar rates. Percentage of PhoP-P relative to the maximum achieved after incubation with wild-type or T438A PhoQ proteins and ATP (1 mM) for the indicated times.
All data correspond to the average ± SEM from three independent experiments. See also Tables S1, S3, and S4 and Figures S2 and S3.

Won-Sik Yeo, et al. Mol Cell. ;45(3):409-421.
7.
Figure 6

Figure 6. Model and Simulations of the Dynamic Behavior of Bifunctional SKs and Sensitivity Analysis of the RR-P Concentration with Respect to the Parameters and Species of the Model. From: Intrinsic Negative Feedback Governs Activation Surge in Two-Component Regulatory Systems.

(A) Model schematics. Reactions between SK states (S) and between RR states (R) are depicted by thick and thin lines, respectively (Table S2). The parameters reflect the reaction rates of the following processes: production of SK (k1), production of RR (k2), ATP binding to the SK (k3), SK autophosphorylation (k4), phosphotransfer from the SK in the kinase state (blue circle) to the RR (k5), ADP dissociation from the SK (k6); ADP-stimulated SK-promoted dephosphorylation of RR-P (green circle) (k9), and the rate of dephosphorylation of the phosphorylated RR in the phosphatase state with ADP remaining bound. An alternative pathway may exist whereby ADP dissociates from the autophosphorylated SK (blue circle) before phosphoryl transfer to the RR takes place. In this case, k7 is the ADP dissociation rate from the SK, and k8 corresponds to phosphotransfer between the SK and the RR. The species amounts and parameter learning process are specified in Table S2 (see parts I–III) and in the Supplemental Information.
(B) Simulation for the behavior of the PhoP/PhoQ TCS. Initially, there are more SK molecules in the kinase state (blue line), which then shift to being mostly in the phosphatase state (green line), resulting in a surge of RR-P (red line). The learned model (red line) correlates well (R2 = 0.9205) with the experimentally determined levels of PhoP-P in vivo (black dots).
(C) Simulation of the behavior of PhoP-P with an SK PhoQ harboring a mutation in the lid that renders it defective for phosphatase activity. Increasing the ADP dissociation rate (parameter k6) by 10-fold predicts sustained levels of the RR-P (red line), and consequently of the mRNA corresponding to the genes regulated by the RR-P.
(D) Sensitivity of the [RR-P] (i.e., concentration of RR-P) to the values of the kinetic parameters presented in Table S2 (see part IV). The values were normalized to facilitate comparisons as described in the Supplemental Experimental Procedures. The most sensitive parameters are: k1 (i.e., the rate by which the SK is produced), k5 (i.e., phosphotransfer to RR activity), k6 (i.e., ADP dissociation from SK), and k9 (i.e., SK phosphatase activity).
(E) Sensitivity of the [RR-P] to the initial values of the different species. The values were normalized as in (A). Most species show limited effects (~± 0.5) on RR-P levels. The values of A0 (i.e., SK concentration) and A1 (i.e., RR concentration) affect both the initial and peak RR-P levels. See also Figures S5 and S6 and Table S2.

Won-Sik Yeo, et al. Mol Cell. ;45(3):409-421.

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