From PII signaling to metabolite sensing: a novel 2-oxoglutarate sensor that details PII-NAGK complex formation.

The widespread PII signal transduction proteins are known for integrating signals of nitrogen and energy supply and regulating cellular behavior by interacting with a multitude of target proteins. The PII protein of the cyanobacterium Synechococcus elongatus forms complexes with the controlling enzyme of arginine synthesis, N-acetyl-L-glutamate kinase (NAGK) in a 2-oxoglutarate- and ATP/ADP-dependent manner. Fusing NAGK and PII proteins to either CFP or YFP yielded a FRET sensor that specifically responded to 2-oxoglutarate. The impact of the fluorescent tags on PII and NAGK was evaluated by enzyme assays, surface plasmon resonance spectroscopy and isothermal calorimetric experiments. The developed FRET sensor provides real-time data on PII - NAGK interaction and its modulation by the effector molecules ATP, ADP and 2-oxoglutarate in vitro. Additionally to its utility to monitor 2-oxoglutarate levels, the FRET assay provided novel insights into PII - NAGK complex formation: (i) It revealed the formation of an encounter-complex between PII and NAGK, which holds the proteins in proximity even in the presence of inhibitors of complex formation; (ii) It revealed that the PII T-loop residue Ser49 is neither essential for complex formation with NAGK nor for activation of the enzyme but necessary to form a stable complex and efficiently relieve NAGK from arginine inhibition; (iii) It showed that arginine stabilizes the NAGK hexamer and stimulates PII - NAGK interaction

Energy Sensing versus 2-Oxoglutarate Dependent ATPase Switch in the Control of Synechococcus PII Interaction with Its Targets NAGK and PipX.

PII proteins constitute a superfamily of highly conserved signaling devices, common in all domains of life. Through binding of the metabolites ATP, ADP and 2-oxoglutarate (2-OG), they undergo conformational changes which allow them to regulate a variety of target proteins including enzymes, transport proteins and transcription factors. But, in reverse, these target proteins also modulate the metabolite sensing properties of PII, as has been recently shown. We used this effect to refine our PII based Förster resonance energy transfer (FRET) sensor and amplify its sensitivity towards ADP. With this enhanced sensor setup we addressed the question whether the PII protein from the model organism Synechococcus elongatus autonomously switches into the ADP conformation through ATPase activity as proposed in a recently published model. The present study disproves ATPase activity as a relevant mechanism for the transition of PII into the ADP state. In the absence of 2-OG, only the ATP/ADP ratio and concentration of ADP directs the competitive interaction of PII with two targets, one of which preferentially binds PII in the ATP-state, the other in the ADP-state

On Mixed Flow Turbines for Automotive Turbocharger Applications

Due to increased demands for improved fuel economy of passenger cars, low-end and part-load performance is of key importance for the design of automotive turbocharger turbines. In an automotive drive cycle, a turbine which can extract more energy at high pressure ratios and lower rotational speeds is desirable. In the literature it is typically found that radial turbines provide peak efficiency at speed ratios of 0.7, but at high pressure ratios and low rotational speeds the blade speed ratio will be low and the rotor will experience high values of positive incidence at the inlet. Based on fundamental considerations, it is shown that mixed flow turbines offer substantial advantages for such applications. Moreover, to prove these considerations an experimental assessment of mixed flow turbine efficiency and optimal blade speed ratio is presented. This has been achieved using a new semi-unsteady measurement approach. Finally, evidence of the benefits of mixed flow turbine behaviour in engine operation is given. Regarding turbocharged engine simulation, the benefit of wide-ranging turbine map measurement data as well as the need for reasonable turbine map extrapolation is illustrated

Measuring the ADP production by P_II.

<p><b>A:</b> P_II-V was incubated with ATP for 1 h at 37°C, then NAGK-C and PipX were added and FRET was measured after 15 min of additional incubation. <b>B:</b> P_II-V was incubated without ATP for 1 h at 37°C, then ATP, NAGK-C and PipX were added and FRET was measured after 15 min of additional incubation. <b>C:</b> P_II-V was incubated without ATP for 1 h at 37°C, then a mixture of ATP, ADP, NAGK-C and PipX was added and FRET was measured after 15 min of additional incubation. Used concentrations: P_II-V, NAGK-C: 0.1 μM, PipX: 1 μM; ATP (A, B): 10 μM (dark blue), 100 μM (blue), 1000 μM (light blue); ATP+ADP mixture (C): 5 μM each (dark blue), 50 μM each (blue) 500 μM each (light blue). Mean values of 3 experiments with standard deviation are shown.</p

Response of the P_II-V NAGK-C FRET sensor towards ADP in the presence of different PipX concentrations.

<p>(A) At a constant ATP concentration of 1 mM the effect of different ADP concentrations on the P_II-V NAGK-C FRET was measured in the absence and presence of different PipX concentrations. P_II-V and NAGK-C were used in concentrations of 0.1 μM. For practical reasons, the data points representing minimum and maximum values of ADP/ATP ratios where in fact derived from measurements without ADP (using the data point at 0.001 mM ADP) or 10 mM ADP without ATP (using the data point 100 mM ADP). Possible trace amounts of contaminating ADP in the ATP solution were not considered here. Mean values of 3 measurements with standard deviation are shown and a sigmoidal dose response curve was fitted using GraphPad Prism 6. (B) The Hill slope and IC₅₀(ADP) values derived from the fitted curves of Fig 1A are presented with error bars indicating the 95% confidence intervals.</p

SPR analysis of the interaction of sensor-chip bound NAGK with P_II or Venus-tagged P_II variants.

<p>His-tagged NAGK protein was immobilized on the surface of flow cell (FC) 2 of a Ni-NTA coated sensor chip; FC 1 served as a control for unspecific binding. P_II-wt (solid line), P_II-ST‑V (long-dashed line), P_II-S49G‑ST (dotted line), P_II-S49G‑ST‑V (dashed line), P_II-E85A-ST‑V (dot-dot-dashed line) and P_II-S49G-E85A-ST‑V (dot-dashed line) were used as analyte. The response difference (∆RU) between FC2 and FC1 during injection and one minute of dissociation is shown.</p

Fusion protein constructs and their FRET-performance.

<p>(<b>A</b>) Schematic representation of NAGK and P_II fusion proteins. Linker domains are denoted “HL” for helical linker, “FL” for flexible linker and “SL” for short linker with the corresponding number of amino acids in brackets. All domains are shown in the same scale. (<b>B</b>) Schematic representation of the assembled complex of FP-tagged P_II and NAGK. Two P_II trimers sandwich one NAGK hexamer. Cerulean domains are shown in blue, Venus domains are shown in yellow. (<b>C</b>) Emission spectra from different combinations of NAGK‑Cerulean and P_II-Venus variants and NAGK‑FL25‑C alone, excited at 433 nm, emission scan from 445 to 640 nm. NAGK‑FL25‑C + P_II-ST‑V in dark red, + P_II-SL4‑V‑ST in red, + P_II-HL15‑V‑ST in light red. NAGK‑HL20‑C + P_II-ST‑V in dark blue, + P_II-SL4‑V‑ST in blue, + P_II-HL15‑V‑ST in light blue. NAGK‑HL25‑C + P_II-ST‑V in dark green, + P_II-SL4‑V‑ST in green, + P_II-HL15‑V‑ST in light green and NAGK‑FL25‑C without P_II in dashed red. All spectra were corrected from background Venus emission and normalized to the Cerulean peak at 475 nm for better comparability. The peaks at 525 nm represent the Venus fluorescence induced by energy transfer from Cerulean. (<b>D</b>) Emission spectra from NAGK‑FL25‑C + P_II-ST‑V in dark red, + P_II-S49G‑ST‑V in green, + P_II-E85A‑ST‑V in light green, + P_II-S49G‑E85A‑ST‑V in blue and NAGK‑FL25‑C without P_II in dashed red. Note that the corrected spectrum recorded with the double mutant P_II-S49G‑E85A‑ST‑V is identical to the spectrum of NAGK‑FL25‑C in the absence of P_II. </p

Influence of effector molecules on the P_II - NAGK complex.

<p>(<b>A</b>) 2-OG induced dissociation of the NAGK‑FL25‑C + P_II-ST‑V complex, as determined by FRET analysis. The FRET ratio (525 nm / 475 nm emission) is background subtracted and normalized to first 6 values. (<b>B</b>) End point measurements of 2-OG induced dissociation. Solid line: NAGK‑FL25‑C and P_II-ST‑V were incubated together for 10 min and then 2-OG was added. After 30 min of incubation the FRET ratio was determined. Dashed line: NAGK‑FL25‑C and P_II-ST‑V were coincubated with 2-OG for 30 min without preincubation and then the FRET ratio was determined. All signals were normalized to values from control experiments without 2-OG. All reactions were performed as triplicates; standard deviation is indicated by error bars. (<b>C</b>) ADP-induced dissociation of the NAGK‑FL25‑C + P_II-ST‑V complex, as determined by FRET analysis. Background subtracted and normalized to first 6 values. 0.1 mM ATP was present in the reaction mix. (<b>D</b>) End point measurements of ADP induced dissociation. Solid line: NAGK‑FL25‑C and P_II-ST‑V were incubated together for 10 min and then ADP was added. After 30 min of incubation the FRET ratio was determined. Dashed line: NAGK‑FL25‑C and P_II-ST‑V were coincubated with ADP for 30 min without preincubation and then the FRET ratio was determined. 0.1 mM ATP was present in the reaction mix, ADP concentrations were ranging from 0.25 to 8 mM. Inset: Magnification showing FRET signals from NAGK‑FL25‑C and P_II-ST‑V in the presence of 2 mM ATP and ADP concentrations ranging from 0.5 to 2 mM (dotted line) representing more physiological concentrations. All signals were normalized to values from control experiments without ADP. All reactions were performed as triplicates; standard deviation is indicated by error bars. (<b>E</b>) Dissociation of NAGK‑FL25‑C and P_II-ST‑V complex with 0.5 mM of malate, fumurate, succinate, oxalacetate, citrate, isocitrate and 2-OG. 0.075 mM ATP was present in all reactions.</p

Association of P_II-V and NAGK-C after P_II-V preincubation with and without ATP, measured by FRET.

<p>P_II-V (0.1 μM) was either preincubated with 10 μM ATP (solid line) or without ATP (dashed line) for 30 min at 37°C. The FRET measurement was started and after 0.5 min NAGK-C (0.1 μM) was added. In the control experiment ATP (10 μM) was added together with NAGK-C.</p