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Discipline
Biological
Keywords
Luciferase
Bioluminescence Resonance Energy Transfer (BRET)
Observation Type
Standalone
Nature
Orphan Data
Submitted
Jan 19th, 2017
Published
May 22nd, 2017
  • Abstract

    Cyclic guanosine monophosphate (cGMP) hydrolyzing phosphodiesterase 5 (PDE5) is multidomain protein, in which cGMP binding to the regulatory GAFa domain allosterically increases the cGMP hydrolytic activity of the catalytic domain. In addition to cyclic nucleotides, GAF domains in phosphodiesterases have been proposed to be regulated by sodium (Na+) ions. The current study was initiated to investigate the effect of Na+ ions on the structure of the GAFa domain of PDE5. Bioluminescence Resonance Energy Transfer (BRET) experiments with various sensor constructs (the GFP2-GAFa-Rluc, GFP2-PDE5-Rluc containing the full-length PDE5, and a control, GFP2-Rluc, construct that does not contain any domains from PDE5) showed a general salt (NaCl and KCl)-induced reduction in the BRET efficiency. Independent determination of Rluc and GFP2 emission of the GFP2-Rluc construct revealed that increased salt concentration enhances Rluc activity without concomitantly increasing GFP2 fluorescence, thus providing a basis for the decrease in the BRET efficiency. The isolated GAFa domain sensor showed similar changes in BRET efficiency with varying concentrations of cGMP at both low (10 mM) and physiologically relevant (100 mM) NaCl concentrations. However, the full-length PDE5 sensor failed to respond to both cGMP and sildenafil at low (10 mM) NaCl concentration. These results suggest a role for NaCl in regulating the structural changes in PDE5, and thus NaCl should be included at 100 mM in BRET assays to detect ligand-induced conformational changes. Additionally, the increase in Rluc activity at higher salt concentrations reported here could be utilized to detect changes in ionic strengths in live cells.

  • Figure
  • Introduction

    Phosphodiesterase 5 is a key regulator of cGMP signaling in a variety of cell types including vascular smooth muscle cells, and in which it functions to control their contractility. It is a multidomain protein with two N-terminally located regulatory GAF domains (GAFa and GAFb) in tandem, and C-terminally located catalytic domain. While the GAFb domain is not known to bind any ligand, the GAFa domain binds cGMP with high specificity, and cGMP binding to the GAFa domain allosterically regulates the activity of the catalytic domain. The structural change induced upon cGMP binding to the GAFa domain has been utilized in developing highly specific, live cell cGMP sensors. The catalytic domain of PDE5 specifically hydrolyzes cGMP, and has been targeted with a number of pharmaceutical inhibitors, including sildenafil, for treatment of diseases such as penile erectile dysfunction and pulmonary hypertension. Structural studies have revealed that the binding of these inhibitors is associated with specific structural changes in the catalytic domain. Ligand binding-induced structural changes in both the GAFa and the catalytic domains in the full-length PDE5 have been extensively explored by developing a Bioluminescence Resonance Energy Transfer (BRET)-based intramolecular conformational sensor.

    BRET is being increasingly utilized for monitoring activation of proteins and signaling pathways in live cells. The underlying mechanism behind BRET is the non-radiative transfer of energy from a bioluminescent donor such as Renilla luciferase (Rluc) to a fluorescent acceptor such as GFP2 (a variant of GFP optimized for excitation by Rluc emission). Key parameters that affect the resonance energy transfer efficiency include the distance between the donor and acceptor, their relative orientation, the quantum yield of the donor and the refractive index of the medium. A typical BRET assay is performed by incubating live cells or cell lysates with the Rluc substrate, and measuring the GFP2 fluorescence and Rluc emission. BRET efficiency is determined as a ratio of GFP2 and Rluc emissions.

  • Objective

    To determine the effect of buffer NaCl concentration on ligand-induced changes in BRET efficiency of PDE5 constructs.

  • Results & Discussion

    Cyclic nucleotide binding to the tandem GAF domains allosterically regulates the activity of the catalytic domain of CyaB1 and CyaB2, adenylyl cyclases from Anabaena. Additionally, Na+ ions have been shown to inhibit the GAF domain-mediated activation of the catalytic domain in these proteins. This inhibitory effect was found to be specific to Na+ ions, and other monovalent cations did not induce such effects on the GAF domain. Importantly, the inhibitory effect of Na+ ion was observed even when the GAF domains from the Anabaena adenylyl cyclase was exchanged for the GAF domains of rat PDE2 indicating the conservation of this mode of regulation across GAF domains. Specific interaction aside, altering the salt concentration will result in an alteration in the ionic strength of the buffer, unless controlled for. An alteration in the ionic strength could affect interactions between amino acids residues in a protein by electrostatic screening leading to changes in the structural properties of the protein. Therefore, an attempt was made to determine the role of Na+ ions on the structure of the cGMP-binding GAFa domain of PDE5. A BRET-based GAFa conformational sensor construct was utilized for this. Lysate prepared from HEK 293T cells transfected with the GFP2-GAFa-Rluc sensor construct was incubated with varying concentrations of NaCl, and intramolecular BRET efficiency (measured as a ratio of GFP2 emission and Rluc emission) was monitored. Increasing NaCl concentration in the buffer resulted in a concentration-dependent reduction in the BRET of the GAFa construct with an effective concentration value for 50% reduction in BRET efficiency (EC50) of 70 ± 43 mM (Fig. 1A). Since BRET efficiency is dependent upon the structure of the protein, this result suggests the possibility of a structural regulation of the GAFa domain by Na+ ions. This was then followed up with experiments with the full-length PDE5 (A2 isoform) domain that contains two GAF domains in tandem. Similar to the isolated GAFa domain, the full-length PDE5 also showed a concentration-dependent reduction in the BRET efficiency with increasing NaCl concentrations with an EC50 value of 218 ± 140 mM (Fig. 1B). However, contrary to the specific inhibitory effect of Na+ seen with the Anabaena proteins, increasing concentrations of KCl also reduced the BRET efficiency (EC50 value of 268 ± 80 mM) suggesting that it could be a general effect of the increase in the salt concentration in the buffer. To confirm this, a construct containing only the GFP2 and Rluc domains, but no domains from PDE5, was used. As seen with the GAFa and full-length PDE5 sensors, the GFP2-Rluc construct also showed a dose-dependent reduction in the BRET efficiency (EC50 values of 95 ± 55 mM and 93 ± 10 mM for NaCl and KCl, respectively) (Fig. 1C). These results suggest that the higher salt concentrations in the buffer negatively impact the energy transfer between Rluc and GFP2.

    The reduction in the BRET efficiency at higher salt concentrations could occur due to either a change in the structure of the proteins resulting in a reduction in the energy transfer or a decrease in the quantum yield of GFP2 protein such that the fluorescence output of GFP2 is reduced, even though a similar amount of non-radiative energy is transferred. To delineate the mechanism behind this, the activity of the Rluc and the fluorescence of the GFP2 proteins in the GFP2-Rluc construct were independently monitored. Increasing the salt concentration of the buffer resulted in a dose-dependent increase in the emission of the Rluc protein with EC50 values of 183 ± 63 mM and 198 ± 62 mM for NaCl and KCl, respectively (Fig. 1D), without significantly impacting the GFP2 emission of the fusion protein (Fig. 1E). Similar increase in the emission was also observed for a construct of the Rluc protein only (EC50 of 154 ± 5 mM) (Fig. 1F). These results suggests that the decrease in the BRET efficiency of the PDE5 constructs is due to an increase in Rluc emission without a concomitant increase in the fluorescence of the GFP2 protein. The increase in the Rluc emission at higher salt concentrations is contrary to the sensitivity of the Firefly luciferase, which is an ATP-consuming enzyme, to higher ionic strengths.

    While the general increase in the Rluc activity observed in the presence of higher salt concentrations is interesting in itself, it is not obvious as to how alterations in the salt concentration would impact the ligand-binding induced conformational change in a sensor protein. Therefore, experiments were performed to understand the impact of NaCl in the ability of BRET-based, PDE5 conformational sensors to detect ligand-induced conformational changes. The salt concentration of 10 mM and 100 mM were chosen based on the EC50 value of BRET efficiency reduction (Fig. 1A). Incubation of lysates prepared from cells expressing the GAFa sensor construct with varying concentrations of cGMP in the presence of low (10 mM) or physiologically relevant (100 mM) NaCl revealed similar increases in the BRET efficiency (EC50 values of 38 ± 3 vs. 22 ± 5 nM at 10 and 100 mM NaCl, respectively). On the other hand, incubation of lysates prepared from cells expressing the full-length PDE5 sensor construct with varying concentrations of cGMP or sildenafil at low (10 mM) NaCl concentration revealed a lack of change in the BRET efficiency of the sensor (Fig. 1H & I), which was seen at 100 mM NaCl concentration (EC50 values of 104 ± 50 and 100 ± 52 nM for cGMP and sildenafil, respectively) (Fig. 1H & I). 

    The lack of change in the BRET efficiency of the full-length PDE5 sensor with cGMP in the presence of low NaCl concentration could be a result of enzymatic hydrolysis of cGMP by the catalytic domain of PDE5. The use of non-hydrolyzable cGMP analogues may thus confirm the effect of salt on PDE5 conformational change. However, experiments described here were performed in the presence of ethylenediaminetetraacetic acid (EDTA), which would chelate metal ions in the buffer, and thus render the catalytic domain of PDE5 inactive. Therefore, the absence of change in BRET efficiency is not a consequence of hydrolysis of cGMP added during the assay. It is also important to note that no change in BRET was observed in the presence of sildenafil citrate at low NaCl concentration. Therefore, the requirement for 100 mM NaCl for the detection of ligand-induced conformational changes in PDE5 suggests a role for Na+ ions by either specific binding to PDE5, or buffer ionic strength in regulating the structure of PDE5. Since the GAFa sensor responded equally well to cGMP in the two NaCl concentrations, it is possible that the GAFb domain in PDE5 is responsible for the sensitivity to NaCl concentration.

  • Conclusions

    The present study is directed towards understanding the effect of NaCl concentration on the structure as well as ligand-induced conformational changes in PDE5-based constructs using the BRET technology. The enzymatic activity of Rluc protein used as a donor in the BRET technology is increased upon an increase in the buffer salt (NaCl as well as KCl) concentration resulting in a net decrease in the BRET efficiency of all GFP2 and Rluc containing sensor constructs tested here. The GAFa domain-based cGMP sensor responds to cGMP at both low as well as physiologically relevant NaCl concentrations. However, the response of the full-length PDE5-based sensor is sensitive to NaCl concentrations, and fails to show changes in BRET efficiency at low NaCl concentrations. Observations reported here will be useful in both designing in vitro conformational sensor assays as well as live cell assays involving changes in intracellular ionic strength.

  • Limitations

    One of the limitations of the present study is the use of crude lysates for determining both the BRET efficiency as well as the luciferase activity. While lysates prepared from transfected cells is experimentally straightforward, the presence of other cellular components could potentially impact the measurements. To alleviate this issue, assays should be performed with purified proteins. Additionally, the effect of increased salt concentrations on the structures of the proteins should be assessed independently using assays such as circular dichroism or hydrogen-deuterium exchange.

  • Alt. Explanations

    The increase in luciferase activities of various sensor constructs reported in the current study is determined at a fixed wavelength. It is possible that the increase in the salinity of the buffer alters the emission spectra of Rluc in a manner similar to the red shift seen in the emission peak of Rluc upon mutation of specific residues or of firefly luciferase in vivo. A full spectral characterization of the Rluc protein at various salt concentrations should be undertaken to determine any shift in the emission spectra. Additionally, while it is assumed that the increase in Rluc emission is due to generic effect of the salt concentration on the structural properties of the protein, it is possible that the increased salt concentration alters certain a specific charge-charge interaction in the protein. Therefore, a structure-guided effort should be undertaken to delineate the mechanism of the increase in Rluc activity at high salt concentrations.

  • Conjectures

    Intracellular ionic strength is tightly controlled in a living cell and an alteration in the intracellular ionic strength could activate specific signaling pathways in the cell such as activation of ion channels or transporters as a counter measure. Indeed, a specific class of proteins have been evolved for the purpose of detecting changes in the intracellular ionic strengths. However, there is a lack of genetically encoded, synthetic reporter for intracellular ionic strength. The sensitive activation of Rluc upon increase in the salt concentration reported here points towards its utilization as a luminescence-based, live cell indicator of changes in the intracellular ionic strength.

  • Methods

    Plasmid constructs

    Previously reported GAFa (GFP2-GAFa-Rluc) and the full-length PDE5 (GFP2-PDE5-Rluc) sensor plasmid constructs have been used here. The GFP2-Rluc and Rluc plasmids were purchased from Perkin-Elmer and used as such.

    Cell culture and transfection

    Human embryonic kidney (HEK) 293T cells were maintained in Dulbecco’s modified Eagle's media (DMEM) with 10% fetal calf serum, 120 mg/L penicillin and 270 mg/L streptomycin at 37°C in a 5% CO2 humidified incubator. Transfections of the cells with specific plasmid DNA were performed with polyethyleneimine lipid according to manufacturers’ protocols.

    BRET assays

    All BRET assays were performed in vitro using the BRET2 assay components i.e. acceptor- GFP2, donor- Rluc and Rluc substrate- Coelenterazine 400a (Molecular Imaging Products). HEK 293T cells transfected with appropriate plasmids were lysed in a buffer of 50 mM HEPES (pH 7.5), containing 2 mM EDTA, 1 mM dithiothreitol, 100 mM NaCl, 10 mM sodium pyrophosphate, 80 µM β-glycerophosphate, 1 mM benzamidine, 1 µg/mL aprotinin, 1 µg/mL leupeptin, 5 µg/mL soybean trypsin inhibitor, 100 µM sodium orthovanadate and 10% glycerol. Cells were lysed by a brief sonication and lysates were centrifuged at 13,000 g for 1 h at 4°C, and the cytosol was collected. Aliquots of the cytosol were incubated with varying concentrations of salts diluted in a buffer containing 50 mM HEPES, pH 7.5 in a total volume of 40 µL at 37°C for 10 min. In experiments to determine the change in the BRET efficiency upon cGMP and sildenafil binding to the GAFa and the PDE5 catalytic domain, lysates were incubated with or without 1 mM cGMP and 100 µM sildenafil citrate under the same conditions. The Rluc substrate coelenterazine 400a (Molecular Imaging Products) was added to a final concentration of 5 µM, and emissions were collected for 0.8 s in a Victor3 microplate reader (Perkin Elmer). Emission filters used for Rluc and GFP2 emission were 410 nm (bandpass 80 nm) and 515 nm (bandpass 30 nm), respectively. BRET was calculated as the ratio of GFP emission per second to Rluc emission per second, and the average of three such measurements is reported.

    Statistical analysis

    All experimental data were analyzed using GraphPad Prism 6, and represent the mean ± S.E.M.

  • Funding statement

    This work was supported by the Department of Biotechnology, Government of India, and a fellowship to KHB from the Council for Scientific and Industrial Research, Government of India.

  • Acknowledgements

    We acknowledge members of the laboratory for useful discussions. KHB acknowledges the Senior Research Fellowship from the Mechanobiology Institute, National University of Singapore, Singapore.

  • Ethics statement

    Not applicable.

  • References
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    Matters10.5/20

    Buffer NaCl concentration regulates Renilla luciferase activity and ligand-induced conformational changes in the BRET-based PDE5 sensor

    Affiliation listing not available.
    Abstractlink

    Cyclic guanosine monophosphate (cGMP) hydrolyzing phosphodiesterase 5 (PDE5) is multidomain protein, in which cGMP binding to the regulatory GAFa domain allosterically increases the cGMP hydrolytic activity of the catalytic domain. In addition to cyclic nucleotides, GAF domains in phosphodiesterases have been proposed to be regulated by sodium (Na+) ions. The current study was initiated to investigate the effect of Na+ ions on the structure of the GAFa domain of PDE5. Bioluminescence Resonance Energy Transfer (BRET) experiments with various sensor constructs (the GFP2-GAFa-Rluc, GFP2-PDE5-Rluc containing the full-length PDE5, and a control, GFP2-Rluc, construct that does not contain any domains from PDE5) showed a general salt (NaCl and KCl)-induced reduction in the BRET efficiency. Independent determination of Rluc and GFP2 emission of the GFP2-Rluc construct revealed that increased salt concentration enhances Rluc activity without concomitantly increasing GFP2 fluorescence, thus providing a basis for the decrease in the BRET efficiency. The isolated GAFa domain sensor showed similar changes in BRET efficiency with varying concentrations of cGMP at both low (10 mM) and physiologically relevant (100 mM) NaCl concentrations. However, the full-length PDE5 sensor failed to respond to both cGMP and sildenafil at low (10 mM) NaCl concentration. These results suggest a role for NaCl in regulating the structural changes in PDE5, and thus NaCl should be included at 100 mM in BRET assays to detect ligand-induced conformational changes. Additionally, the increase in Rluc activity at higher salt concentrations reported here could be utilized to detect changes in ionic strengths in live cells.

    Figurelink

    Figure 1. Effect of salt (NaCl or KCl) on the conformational change in BRET-based PDE5 sensors.

    (A-C) Graphs showing the BRET efficiencies (ratio of GFP2 and Rluc emission at 37°C) determined from lysates prepared from cells expressing the GAFa (A), the full-length PDE5 (B) and the GFP2-Rluc (C) sensor constructs at various salt concentrations. Note the concentration-dependent decrease in the BRET efficiency at higher salt concentrations. Insets in each panel show a plot of EC50 values for individual constructs.

    (D) Graph showing the luciferase emission (counts per second; CPS) of lysates prepared from cells expressing the GFP2-Rluc sensor construct at various salt concentrations. Inset shows a plot of EC50 values.

    (E) Graph showing the fluorescence of lysates prepared from cells expressing the GFP2-Rluc sensor construct at various salt concentrations.

    (F) Graph showing the luciferase activity of lysates prepared from cells expressing the Rluc protein at various salt concentrations. Inset shows a plot of EC50 values.

    (G) Graph showing the percentage change in the BRET efficiency of lysates prepared from cells expressing the GAFa sensor construct with increasing concentrations of cGMP at low (10 mM) and relevant (100 mM) NaCl concentrations. Inset shows a plot of EC50 values.

    (H & I) Graph showing the percentage change in the BRET efficiency of lysates prepared from cells expressing the PDE5 sensor construct with increasing concentrations of cGMP (H) and sildenafil (I) at low (10 mM) and relevant (100 mM) NaCl concentrations. Insets show a plot of EC50 values (ND, could not be determined). Data shown are mean ± S.E.M of a minimum of two measurements from a representative experiment.

    Introductionlink

    Phosphodiesterase 5 is a key regulator of cGMP signaling in a variety of cell types including vascular smooth muscle cells, and in which it functions to control their contractility[1]. It is a multidomain protein with two N-terminally located regulatory GAF domains (GAFa and GAFb) in tandem, and C-terminally located catalytic domain. While the GAFb domain is not known to bind any ligand, the GAFa domain binds cGMP with high specificity[2], and cGMP binding to the GAFa domain allosterically regulates the activity of the catalytic domain[3][4]. The structural change induced upon cGMP binding to the GAFa domain has been utilized in developing highly specific, live cell cGMP sensors[2][5][6]. The catalytic domain of PDE5 specifically hydrolyzes cGMP[7][8], and has been targeted with a number of pharmaceutical inhibitors, including sildenafil, for treatment of diseases such as penile erectile dysfunction and pulmonary hypertension[9][10]. Structural studies have revealed that the binding of these inhibitors is associated with specific structural changes in the catalytic domain[11]. Ligand binding-induced structural changes in both the GAFa and the catalytic domains in the full-length PDE5 have been extensively explored by developing a Bioluminescence Resonance Energy Transfer (BRET)-based intramolecular conformational sensor[12].

    BRET is being increasingly utilized for monitoring activation of proteins and signaling pathways in live cells[13][14][15]. The underlying mechanism behind BRET is the non-radiative transfer of energy from a bioluminescent donor such as Renilla luciferase (Rluc) to a fluorescent acceptor such as GFP2 (a variant of GFP optimized for excitation by Rluc emission)[16]. Key parameters that affect the resonance energy transfer efficiency include the distance between the donor and acceptor, their relative orientation, the quantum yield of the donor and the refractive index of the medium[17]. A typical BRET assay is performed by incubating live cells or cell lysates with the Rluc substrate, and measuring the GFP2 fluorescence and Rluc emission. BRET efficiency is determined as a ratio of GFP2 and Rluc emissions.

    Objectivelink

    To determine the effect of buffer NaCl concentration on ligand-induced changes in BRET efficiency of PDE5 constructs.

    Results & Discussionlink

    Cyclic nucleotide binding to the tandem GAF domains allosterically regulates the activity of the catalytic domain of CyaB1 and CyaB2, adenylyl cyclases from Anabaena[18][19]. Additionally, Na+ ions have been shown to inhibit the GAF domain-mediated activation of the catalytic domain in these proteins[20]. This inhibitory effect was found to be specific to Na+ ions, and other monovalent cations did not induce such effects on the GAF domain[20]. Importantly, the inhibitory effect of Na+ ion was observed even when the GAF domains from the Anabaena adenylyl cyclase was exchanged for the GAF domains of rat PDE2 indicating the conservation of this mode of regulation across GAF domains[20]. Specific interaction aside, altering the salt concentration will result in an alteration in the ionic strength of the buffer, unless controlled for. An alteration in the ionic strength could affect interactions between amino acids residues in a protein by electrostatic screening[21][22] leading to changes in the structural properties of the protein[23]. Therefore, an attempt was made to determine the role of Na+ ions on the structure of the cGMP-binding GAFa domain of PDE5. A BRET-based GAFa conformational sensor construct was utilized for this[2]. Lysate prepared from HEK 293T cells transfected with the GFP2-GAFa-Rluc sensor construct was incubated with varying concentrations of NaCl, and intramolecular BRET efficiency (measured as a ratio of GFP2 emission and Rluc emission) was monitored[2]. Increasing NaCl concentration in the buffer resulted in a concentration-dependent reduction in the BRET of the GAFa construct with an effective concentration value for 50% reduction in BRET efficiency (EC50) of 70 ± 43 mM (Fig. 1A). Since BRET efficiency is dependent upon the structure of the protein[12][24], this result suggests the possibility of a structural regulation of the GAFa domain by Na+ ions. This was then followed up with experiments with the full-length PDE5 (A2 isoform) domain that contains two GAF domains in tandem[1]. Similar to the isolated GAFa domain, the full-length PDE5 also showed a concentration-dependent reduction in the BRET efficiency with increasing NaCl concentrations with an EC50 value of 218 ± 140 mM (Fig. 1B). However, contrary to the specific inhibitory effect of Na+ seen with the Anabaena proteins[20], increasing concentrations of KCl also reduced the BRET efficiency (EC50 value of 268 ± 80 mM) suggesting that it could be a general effect of the increase in the salt concentration in the buffer. To confirm this, a construct containing only the GFP2 and Rluc domains, but no domains from PDE5, was used. As seen with the GAFa and full-length PDE5 sensors, the GFP2-Rluc construct also showed a dose-dependent reduction in the BRET efficiency (EC50 values of 95 ± 55 mM and 93 ± 10 mM for NaCl and KCl, respectively) (Fig. 1C). These results suggest that the higher salt concentrations in the buffer negatively impact the energy transfer between Rluc and GFP2.

    The reduction in the BRET efficiency at higher salt concentrations could occur due to either a change in the structure of the proteins resulting in a reduction in the energy transfer or a decrease in the quantum yield of GFP2 protein such that the fluorescence output of GFP2 is reduced, even though a similar amount of non-radiative energy is transferred[17]. To delineate the mechanism behind this, the activity of the Rluc and the fluorescence of the GFP2 proteins in the GFP2-Rluc construct were independently monitored. Increasing the salt concentration of the buffer resulted in a dose-dependent increase in the emission of the Rluc protein with EC50 values of 183 ± 63 mM and 198 ± 62 mM for NaCl and KCl, respectively (Fig. 1D), without significantly impacting the GFP2 emission of the fusion protein (Fig. 1E). Similar increase in the emission was also observed for a construct of the Rluc protein only (EC50 of 154 ± 5 mM) (Fig. 1F). These results suggests that the decrease in the BRET efficiency of the PDE5 constructs is due to an increase in Rluc emission without a concomitant increase in the fluorescence of the GFP2 protein. The increase in the Rluc emission at higher salt concentrations is contrary to the sensitivity of the Firefly luciferase, which is an ATP-consuming enzyme, to higher ionic strengths[25].

    While the general increase in the Rluc activity observed in the presence of higher salt concentrations is interesting in itself, it is not obvious as to how alterations in the salt concentration would impact the ligand-binding induced conformational change in a sensor protein. Therefore, experiments were performed to understand the impact of NaCl in the ability of BRET-based, PDE5 conformational sensors[2][12] to detect ligand-induced conformational changes. The salt concentration of 10 mM and 100 mM were chosen based on the EC50 value of BRET efficiency reduction (Fig. 1A). Incubation of lysates prepared from cells expressing the GAFa sensor construct with varying concentrations of cGMP in the presence of low (10 mM) or physiologically relevant (100 mM) NaCl revealed similar increases in the BRET efficiency (EC50 values of 38 ± 3 vs. 22 ± 5 nM at 10 and 100 mM NaCl, respectively). On the other hand, incubation of lysates prepared from cells expressing the full-length PDE5 sensor construct with varying concentrations of cGMP or sildenafil at low (10 mM) NaCl concentration revealed a lack of change in the BRET efficiency of the sensor (Fig. 1H & I), which was seen at 100 mM NaCl concentration (EC50 values of 104 ± 50 and 100 ± 52 nM for cGMP and sildenafil, respectively) (Fig. 1H & I). 

    The lack of change in the BRET efficiency of the full-length PDE5 sensor with cGMP in the presence of low NaCl concentration could be a result of enzymatic hydrolysis of cGMP by the catalytic domain of PDE5. The use of non-hydrolyzable cGMP analogues may thus confirm the effect of salt on PDE5 conformational change. However, experiments described here were performed in the presence of ethylenediaminetetraacetic acid (EDTA), which would chelate metal ions in the buffer, and thus render the catalytic domain of PDE5 inactive. Therefore, the absence of change in BRET efficiency is not a consequence of hydrolysis of cGMP added during the assay. It is also important to note that no change in BRET was observed in the presence of sildenafil citrate at low NaCl concentration. Therefore, the requirement for 100 mM NaCl for the detection of ligand-induced conformational changes in PDE5 suggests a role for Na+ ions by either specific binding to PDE5, or buffer ionic strength in regulating the structure of PDE5. Since the GAFa sensor responded equally well to cGMP in the two NaCl concentrations, it is possible that the GAFb domain in PDE5 is responsible for the sensitivity to NaCl concentration.

    Conclusionslink

    The present study is directed towards understanding the effect of NaCl concentration on the structure as well as ligand-induced conformational changes in PDE5-based constructs using the BRET technology. The enzymatic activity of Rluc protein used as a donor in the BRET technology is increased upon an increase in the buffer salt (NaCl as well as KCl) concentration resulting in a net decrease in the BRET efficiency of all GFP2 and Rluc containing sensor constructs tested here. The GAFa domain-based cGMP sensor responds to cGMP at both low as well as physiologically relevant NaCl concentrations. However, the response of the full-length PDE5-based sensor is sensitive to NaCl concentrations, and fails to show changes in BRET efficiency at low NaCl concentrations. Observations reported here will be useful in both designing in vitro conformational sensor assays[2][12][24][26] as well as live cell assays involving changes in intracellular ionic strength[27][28].

    Limitationslink

    One of the limitations of the present study is the use of crude lysates for determining both the BRET efficiency as well as the luciferase activity. While lysates prepared from transfected cells is experimentally straightforward, the presence of other cellular components could potentially impact the measurements. To alleviate this issue, assays should be performed with purified proteins. Additionally, the effect of increased salt concentrations on the structures of the proteins should be assessed independently using assays such as circular dichroism[20] or hydrogen-deuterium exchange[26].

    Alternative Explanationslink

    The increase in luciferase activities of various sensor constructs reported in the current study is determined at a fixed wavelength. It is possible that the increase in the salinity of the buffer alters the emission spectra of Rluc in a manner similar to the red shift seen in the emission peak of Rluc upon mutation of specific residues[29] or of firefly luciferase in vivo[30]. A full spectral characterization of the Rluc protein at various salt concentrations should be undertaken to determine any shift in the emission spectra. Additionally, while it is assumed that the increase in Rluc emission is due to generic effect of the salt concentration on the structural properties of the protein, it is possible that the increased salt concentration alters certain a specific charge-charge interaction in the protein. Therefore, a structure-guided effort should be undertaken to delineate the mechanism of the increase in Rluc activity at high salt concentrations.

    Conjectureslink

    Intracellular ionic strength is tightly controlled in a living cell and an alteration in the intracellular ionic strength could activate specific signaling pathways in the cell such as activation of ion channels or transporters as a counter measure[27][31]. Indeed, a specific class of proteins have been evolved for the purpose of detecting changes in the intracellular ionic strengths[28]. However, there is a lack of genetically encoded, synthetic reporter for intracellular ionic strength. The sensitive activation of Rluc upon increase in the salt concentration reported here points towards its utilization as a luminescence-based, live cell indicator of changes in the intracellular ionic strength.

    Methodslink

    Plasmid constructs

    Previously reported GAFa (GFP2-GAFa-Rluc)[2] and the full-length PDE5 (GFP2-PDE5-Rluc)[12] sensor plasmid constructs have been used here. The GFP2-Rluc and Rluc plasmids were purchased from Perkin-Elmer and used as such.

    Cell culture and transfection

    Human embryonic kidney (HEK) 293T cells were maintained in Dulbecco’s modified Eagle's media (DMEM) with 10% fetal calf serum, 120 mg/L penicillin and 270 mg/L streptomycin at 37°C in a 5% CO2 humidified incubator. Transfections of the cells with specific plasmid DNA were performed with polyethyleneimine lipid according to manufacturers’ protocols.

    BRET assays

    All BRET assays were performed in vitro using the BRET2 assay components i.e. acceptor- GFP2, donor- Rluc and Rluc substrate- Coelenterazine 400a (Molecular Imaging Products)[2]. HEK 293T cells transfected with appropriate plasmids were lysed in a buffer of 50 mM HEPES (pH 7.5), containing 2 mM EDTA, 1 mM dithiothreitol, 100 mM NaCl, 10 mM sodium pyrophosphate, 80 µM β-glycerophosphate, 1 mM benzamidine, 1 µg/mL aprotinin, 1 µg/mL leupeptin, 5 µg/mL soybean trypsin inhibitor, 100 µM sodium orthovanadate and 10% glycerol. Cells were lysed by a brief sonication and lysates were centrifuged at 13,000 g for 1 h at 4°C, and the cytosol was collected. Aliquots of the cytosol were incubated with varying concentrations of salts diluted in a buffer containing 50 mM HEPES, pH 7.5 in a total volume of 40 µL at 37°C for 10 min. In experiments to determine the change in the BRET efficiency upon cGMP and sildenafil binding to the GAFa and the PDE5 catalytic domain, lysates were incubated with or without 1 mM cGMP and 100 µM sildenafil citrate under the same conditions. The Rluc substrate coelenterazine 400a (Molecular Imaging Products) was added to a final concentration of 5 µM, and emissions were collected for 0.8 s in a Victor3 microplate reader (Perkin Elmer). Emission filters used for Rluc and GFP2 emission were 410 nm (bandpass 80 nm) and 515 nm (bandpass 30 nm), respectively. BRET was calculated as the ratio of GFP emission per second to Rluc emission per second, and the average of three such measurements is reported.

    Statistical analysis

    All experimental data were analyzed using GraphPad Prism 6, and represent the mean ± S.E.M.

    Funding Statementlink

    This work was supported by the Department of Biotechnology, Government of India, and a fellowship to KHB from the Council for Scientific and Industrial Research, Government of India.

    Acknowledgementslink

    We acknowledge members of the laboratory for useful discussions. KHB acknowledges the Senior Research Fellowship from the Mechanobiology Institute, National University of Singapore, Singapore.

    Conflict of interestlink

    The authors declare no conflicts of interest.

    Ethics Statementlink

    Not applicable.

    No fraudulence is committed in performing these experiments or during processing of the data. We understand that in the case of fraudulence, the study can be retracted by ScienceMatters.

    Referenceslink
    1. Marco Conti, Joseph Beavo
      Biochemistry and Physiology of Cyclic Nucleotide Phosphodiesterases: Essential Components in Cyclic Nucleotide Signaling
      Annual Review of Biochemistry, 76/2007, pages 481-511 chrome_reader_mode
    2. Kabir Hassan Biswas, Shailaja Sopory, Sandhya S. Visweswariah
      The GAF Domain of the cGMP-Binding, cGMP-Specific Phosphodiesterase (PDE5) Is a Sensor and a Sink for cGMP
      Biochemistry, 47/2008, pages 3534-3543 chrome_reader_mode
    3. Jackie D. Corbin, Mitsi A. Blount, James L. Weeks, Alfreda Beasley, Karl P. Kuhn, Yew S. J. Ho, Layla F. Saidi, James H. Hurley, Jun Kotera, Sharron H. Francis
      [3H]Sildenafil Binding to Phosphodiesterase-5 Is Specific, Kinetically Heterogeneous, and Stimulated by cGMP
      Molecular Pharmacology, 63/2003, pages 1364-1372 chrome_reader_mode
    4. Irina G. Rybalkina, Xiao-Bo Tang, Sergei D. Rybalkin
      Multiple Affinity States of cGMP-Specific Phosphodiesterase for Sildenafil Inhibition Defined by cGMP-Dependent and cGMP-Independent Mechanisms
      Molecular Pharmacology, 77/2010, pages 670-677 chrome_reader_mode
    5. Viacheslav O Nikolaev, Stepan Gambaryan, Martin J Lohse
      Fluorescent sensors for rapid monitoring of intracellular cGMP
      Nature Methods, 3/2006, pages 23-25 chrome_reader_mode
    6. Michael Russwurm, Florian Mullershausen, Andreas Friebe, Ronald Jäger, Corina Russwurm, Doris Koesling
      Design of fluorescence resonance energy transfer (FRET)-based cGMP indicators: a systematic approach
      Biochemical Journal, 407/2007, pages 69-77 chrome_reader_mode
    7. Illarion V. Turko, Sharron H. Francis, Jackie D. Corbin
      Studies of the Molecular Mechanism of Discrimination between cGMP and cAMP in the Allosteric Sites of the cGMP-binding cGMP-specific Phosphodiesterase (PDE5)
      The Journal of Biological Chemistry, 274/1999, pages 29038-29041 chrome_reader_mode
    8. Kam Y.J Zhang, Graeme L Card, Yoshihisa Suzuki,more_horiz, Gideon Bollag
      A Glutamine Switch Mechanism for Nucleotide Selectivity by Phosphodiesterases
      Molecular Cell, 15/2004, pages 279-286 chrome_reader_mode
    9. David P. Rotella
      Phosphodiesterase 5 inhibitors: current status and potential applications
      Nature Reviews Drug Discovery, 1/2002, pages 674-682 chrome_reader_mode
    10. Galiè, Nazzareno, Ghofrani,more_horiz, Gérald
      Sildenafil Citrate Therapy for Pulmonary Arterial Hypertension
      New England Journal of Medicine, 353/2005, pages 2148-2157 chrome_reader_mode
    11. Huanchen Wang, Yudong Liu, Qing Huai, Jiwen Cai, Roya Zoraghi, Sharron H. Francis, Jackie D. Corbin, Howard Robinson, Zhongcheng Xin, Guiting Lin, Hengming Ke
      Multiple Conformations of Phosphodiesterase-5: IMPLICATIONS FOR ENZYME FUNCTION AND DRUG DEVELOPMENT
      The Journal of Biological Chemistry, 281/2006, pages 21469-21479 chrome_reader_mode
    12. Kabir H. Biswas, Sandhya S. Visweswariah
      Distinct Allostery Induced in the Cyclic GMP-binding, Cyclic GMP-specific Phosphodiesterase (PDE5) by Cyclic GMP, Sildenafil, and Metal Ions
      The Journal of Biological Chemistry, 286/2011, pages 8545-8554 chrome_reader_mode
    13. Kevin D.G. Pfleger, Jasmin R. Dromey, Matthew B. Dalrymple,more_horiz, Karin A. Eidne
      Extended bioluminescence resonance energy transfer (eBRET) for monitoring prolonged protein–protein interactions in live cells
      Cellular Signalling, 18/2006, pages 1664-1670 chrome_reader_mode
    14. Kevin D G Pfleger, Karin A Eidne
      Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET)
      Nature Methods, 3/2006, pages 165-174 chrome_reader_mode
    15. Jie Yang, Derrick Cumberbatch, Samuel Centanni, Shu-Qun Shi, Danny Winder, Donna Webb, Carl Hirschie Johnson
      Coupling optogenetic stimulation with NanoLuc-based luminescence (BRET) Ca++ sensing
      Nature Communications, 7/2016, page 13268 chrome_reader_mode
    16. Abhijit de, Andreas Markus Loening, Sanjiv Sam Gambhir
      An Improved Bioluminescence Resonance Energy Transfer Strategy for Imaging Intracellular Events in Single Cells and Living Subjects
      Cancer Research, 67/2007, pages 7175-7183 chrome_reader_mode
    17. Madan Rao, Satyajit Mayor
      Use of Forster's resonance energy transfer microscopy to study lipid rafts
      Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1746/2005, pages 221-233 chrome_reader_mode
    18. Sandra Bruder, Jürgen U. Linder, Sergio E. Martinez, Ning Zheng, Joseph A. Beavo, Joachim E. Schultz
      The cyanobacterial tandem GAF domains from the cyaB2 adenylyl cyclase signal via both cAMP-binding sites
      Proceedings of the National Academy of Sciences, 102/2005, pages 3088-3092 chrome_reader_mode
    19. Tobias Kanacher, Anita Schultz, Jürgen U. Linder, Joachim E. Schultz
      A GAF‐domain‐regulated adenylyl cyclase from Anabaena is a self‐activating cAMP switch
      The EMBO Journal, 21/2002, pages 3672-3680 chrome_reader_mode
    20. Martin Cann
      A subset of GAF domains are evolutionarily conserved sodium sensors
      Molecular Microbiology, 64/2007, pages 461-472 chrome_reader_mode
    21. James B. Matthew
      Electrostatic Effects in Proteins
      Annual Review of Biophysics and Biophysical Chemistry, 14/1985, pages 387-417 chrome_reader_mode
    22. Jan J. Spitzer, Bert Poolman
      Electrochemical structure of the crowded cytoplasm
      Trends in Biochemical Sciences, 30/2005, pages 536-541 chrome_reader_mode
    23. Qian Wang, Kao-Chen Liang, Arkadiusz Czader, M. Neal Waxham, Margaret S. Cheung
      The Effect of Macromolecular Crowding, Ionic Strength and Calcium Binding on Calmodulin Dynamics
      PLOS Computational Biology, 7/2011, page e1002114 chrome_reader_mode
    24. Subhalaxmi Nambi, Suguna Badireddy, Sandhya S. Visweswariah, Ganesh S. Anand
      Cyclic AMP-induced Conformational Changes in Mycobacterial Protein Acetyltransferases
      The Journal of Biological Chemistry, 287/2012, pages 18115-18129 chrome_reader_mode
    25. Arne Lundin
      Optimization of the Firefly Luciferase Reaction for Analytical Purposes
      Advances in Biochemical Engineering/Biotechnology: Bioluminescence: Fundamentals and Applications in Biotechnology - Volume 2 , 145/2014, pages 31-62 chrome_reader_mode
    26. Kabir Hassan Biswas​, Suguna Badireddy, Abinaya Rajendran, Ganesh Srinivasan Anand, Sandhya S. Visweswariah​
      Cyclic nucleotide binding and structural changes in the isolated GAF domain of Anabaena adenylyl cyclase, CyaB2
      PeerJ, 3/2015, page e882 chrome_reader_mode
    27. Thomas Voets, Guy Droogmans, Gert Raskin, Jan Eggermont, Bernd Nilius
      Reduced intracellular ionic strength as the initial trigger for activation of endothelial volume-regulated anion channels
      Proceedings of the National Academy of Sciences, 96/1999, pages 5298-5303 chrome_reader_mode
    28. Esther Biemans-Oldehinkel, Nik A. B. N. Mahmood, Bert Poolman
      A sensor for intracellular ionic strength
      Proceedings of the National Academy of Sciences, 103/2006, pages 10624-10629 chrome_reader_mode
    29. Andreas Markus Loening, Anna M Wu, Sanjiv Sam Gambhir
      Red-shifted Renilla reniformis luciferase variants for imaging in living subjects
      Nature Methods, 4/2007, pages 641-643 chrome_reader_mode
    30. Hui Zhao, Timothy C. Doyle, Olivier Coquoz, Flora Kalish, Bradley W. Rice, Christopher H. Contag
      Emission spectra of bioluminescent reporters and interaction with mammalian tissue determine the sensitivity of detection in vivo
      Journal of Biomedical Optics, 10/2005, page 041210 chrome_reader_mode
    31. Carolyn L. Cannon, Srisaila Basavappa, Kevin Strange
      Intracellular ionic strength regulates the volume sensitivity of a swelling-activated anion channel
      American Journal of Physiology-Cell Physiology, 275/1998, pages C416-C422 chrome_reader_mode
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