We report and demonstrate an optimized nitrocellulose binding assay that can be used to quantify autophosphorylation of purified bacterial histidine kinases. Our method has several advantages over traditional SDS-PAGE based techniques, providing a valuable alternative for characterizing these important proteins.
We demonstrate a useful method for quantifying autophosphorylation of purified bacterial histidine kinases. Histidine kinases are known for their involvement in two-component signal transduction, a ubiquitous system through which bacteria sense and respond to environmental stimuli. Two-component signaling features autophosphorylation of a histidine kinase, followed by phosphotransfer to the receiver domain of a response regulator protein, which ultimately leads to an output response. Autophosphorylation of the histidine kinase is responsive to the presence of a cognate environmental stimulus, thereby giving bacteria a means to detect and respond to changes in the environment. Despite their importance in bacterial biology, histidine kinases remain poorly understood due to the inherent lability of phosphohistidine. Conventional methods for studying these proteins, such as SDS-PAGE autoradiography, have significant shortcomings. We have developed a nitrocellulose binding assay that can be used to characterize histidine kinases. The protocol for this assay is simple and easy to execute. Our method is higher throughput, less time-consuming, and offers a greater dynamic range than SDS-PAGE autoradiography.
Adaptive response is crucial for bacterial survival. In order to detect and respond to environmental changes, bacteria use a stimulus-response system known as two-component signaling.1,2 In a typical two-component system, the histidine kinase detects a cognate stimulus, autophosphorylates its conserved histidine residue, then transfers phosphate to a conserved aspartate residue on the receiver domain of a response regulator protein.3 This event triggers a change in the activity of the response regulator, which stimulates a downstream effect.4,5 Thus, bacteria are able to sense and adapt to changes in the local environment. Some two-component signaling systems deviate from this archetype. In some cases, the sensory domain of the histidine kinase is a stand-alone protein, which directly detects the sensory input and modifies kinase activity through a protein-protein interaction.6–8 However, the fundamental process and overall role of the system remains the same. Two-component signaling is a ubiquitous stimulus-response system that is essential for bacterial survival, and histidine kinases play a critical role in the transduction of the signal.9
Despite the importance of histidine kinases to bacterial biology, they remain poorly characterized. This is due to the inherent instability of phosphohistidine, and the lack of a practical method for measuring autophosphorylation. Phosphohistidine is more labile than phosphoserine, phosphothreonine, and phosphotyrosine.10 Thus, techniques that are commonly used to analyze Ser/Thr/Tyr kinases are not applicable for histidine kinases.11 In vitro assays to study histidine kinases have largely been limited to SDS-PAGE autoradiography.12,13 In this method, [γ-32P]-ATP is incubated with the kinase, and phosphorylation of the kinase is analyzed by polyacrylamide gel electrophoresis (PAGE) followed by autoradiography of the gel. This method can be used to monitor kinase autophosphorylation, as well as phosphotransfer from the kinase to a response regulator. However, this method has notable shortcomings. PAGE-based assays are low throughput and time-consuming. Such limitations are not conducive to characterizing a protein and ascertaining its kinetic parameters. An alternative method for studying histidine kinases that was published recently utilizes phosphohistidine antibodies to detect autophosphorylation.14 While this method has the advantage of distinguishing between 1-phosphohistidine and 3-phosphohistidine, depending on the instrumentation used for detection, this method may not offer a large dynamic range or high upper limit of detection. Thus, there is a need for a faster, less laborious, and more sensitive assay that can be used to study these important proteins.
Here, we describe and demonstrate a carefully developed nitrocellulose binding assay that can be used to quantify autophosphorylation of purified bacterial histidine kinases in vitro. This assay is higher throughput and less time-consuming than PAGE-based assays. The method also utilizes Cherenkov radiation for phosphohistidine quantification, which offers a high upper limit of detection and a large dynamic range. The assay can be used to determine kinetic parameters for histidine kinases.
Caution: This protocol requires appropriate training in the use and handling of radioactive materials. Please use the requisite personal protective equipment when performing this assay, including beta radiation shielding. Radioactive waste must be handled carefully, as large volumes of waste are generated during the washing phase of the experiment. Ensure that the waste is stored in an upright container such as a large bucket or bottle that will not be easily knocked over or spilled. Once the experiment is concluded, carefully transfer all liquid waste to appropriately marked radioactive waste containers. Handle all materials with care, and keep a Geiger counter nearby to monitor the workspace for contamination.
NOTE: This protocol is a revised version of a previously reported assay from our group.15 Phosphohistidine stability in H3PO4 should be tested for any uncharacterized histidine kinase prior to using this method. The phosphohistidine stability test has been described previously.15 A negative control that does not contain kinase must be included. This is necessary in order to subtract the background signal from each sample, and ensure that [γ-32P]-ATP is sufficiently washed from the membrane.
1. Preparation of Reagents and Materials
2. Reaction Initiation and Quenching
3. Spotting of Quenched Reactions on Nitrocellulose
4. Nitrocellulose Processing
5. Exposure to Storage Phosphor Screen
Note: This section is optional. Exposing the membrane to a phosphor screen is beneficial in that it allows for visualization of the intensity of radiolabeled kinase in each spot on the membrane. The relative intensity of these spots is directly proportional to the amount of phosphorylated histidine kinase in each spot. The intensity can be quantified with image processing software, and these results can be compared to those generated from section 7. Furthermore, this step allows for quality control. Abnormalities seen in this scan might explain irregular results obtained from scintillation counting.
6. Preparing Nitrocellulose Membrane for Scintillation Counting
7. Scintillation Counting
A representative data set was generated featuring an image captured with a phosphor imager (Figure 1), Ponceau S stain of the nitrocellulose membrane (Figure 2), and scintillation counting data (Figure 3). Figure 3A shows the enzyme kinetic constants in a Lineweaver-Burk plot. These results were obtained using a purified histidine kinase from the gram-negative species Vibrio parahaemolyticus (gene ID 1189383). The protocol used to purify this kinase is described in section 1.1. The final kinase concentration in the reaction was 7.5 µM. The final ATP concentrations ranged from 0 µM to 1.28 mM. The [γ-32P]-ATP solution was 171.97 CPM/pmol ATP. These data can all be generated in a single day using the procedure we have described.
Figure 1: Ponceau S staining of a nitrocellulose membrane. Ponceau stain showing the kinase bound to the nitrocellulose membrane. The membrane was spotted with reactions containing various concentrations of ATP, but constant kinase concentration. No protein is detected in the no-kinase control spots (4th and 8th row). Ponceau S staining solution was composed of 0.1% Ponceau S (w/v) in 5% acetic acid. Please click here to view a larger version of this figure.
Figure 2: Fluorography results. Scanned image of a phosphor screen that had been exposed to a nitrocellulose membrane. The membrane was spotted with reactions containing constant kinase concentration, and various concentrations of ATP. Each concentration was assayed in triplicate. At each concentration, a no kinase control was included (4th and 8th row) to demonstrate that radiolabeled ATP is completely removed from the membrane during the washing step. Please click here to view a larger version of this figure.
Figure 3: Scintillation counting data. A) Double-reciprocal plot of the autophosphorylation of histidine kinase. The substrate-dependence curve was generated from scintillation counting. Phosphohistidine formation was calculated using the slope of Figure 3B (CPM/pmol ATP), which was also generated with scintillation spectrometry. B) Standard curve of the CPM/pmol of ATP. The slope is used to calculate the amount of phosphohistidine (pmol) present in each sample. Please click here to view a larger version of this figure.
The nitrocellulose binding assay we have described has many advantages over previously used methods to characterize histidine kinases. In comparison to traditional SDS/PAGE-based autoradiography, our method is higher throughput and less time-consuming. The nitrocellulose membrane is easier to handle than SDS gels, and does not need to be fixed. Ponceau staining the nitrocellulose allows for the protein spots to be visualized. This provides an easy way to cut out each spot for scintillation counting, and determine that protein loading is consistent across all spots. Scintillation counting of each spot provides accurate results that can be easily converted to reaction velocity using the slope of the standard curve shown in Figure 3B.
Although exposing the nitrocellulose membrane to a storage phosphor screen adds time to the total duration of the experiment, this step offers insight into the success of the blotting. Any discrepancies that are noticed in the scintillation counting data can potentially be explained by the complementary phosphor image. If, for instance, the membrane was not sufficiently washed, the phosphor image will reveal this information. Fluorography of the membrane is recommended to anyone who plans to use this assay, as the results from this step can be a valuable piece of supplemental information.
Another significant advantage of this assay is the ability to generate data from scintillation counting. Cutting out each individual spot for scintillation counting is preferable to use of image processing software to quantify band intensity, as is typically done for PAGE-based assays. The upper limit and dynamic range for detection are far higher with scintillation counting. Generating a standard curve to determine the CPM/pmol ATP make it easy to calculate the rate of autophosphorylation from the raw data. Using this method, we are able to accurately detect picomoles of phosphorylated product.
Despite the many advantages of using our method, it is not without its limitations. Our method does not distinguish between 1-phosphohistidine and 3-phosphohistidine. Although phosphohistidine antibodies can be used to distinguish between these phosphorylated products, our method still has the advantage of utilizing Cherenkov radiation to quantify phosphorylation. Cherenkov radiation provides a large dynamic range and high upper limit of detection. Depending on the method of detection, using antibodies to quantify phosphohistidine formation may suffer from a lesser upper limit and dynamic range. Our method is also limited to being used with purified proteins, and can't be used to detect phosphorylated kinases in vivo. Our method requires radiolabeled substrate, which may deter labs that are not equipped for radioactivity. Ultimately, this method is mostly suitable for labs that are already equipped to handle radioactivity, and are interested in a higher throughput and less time consuming alternative to frequently used SDS-PAGE techniques to study histidine kinases. Those who are studying Ser/Thr/Tyr kinases may also find this method to be useful, despite the relative abundance of alternative methods for studying these proteins.
Characterization of histidine kinases has long been elusive despite advances in methods to quantify other types of kinases. With this assay, we may begin to characterize these biologically relevant yet poorly understood proteins. This advancement in methodology for quantifying histidine kinase autophosphorylation will ultimately improve our understanding of two-component signaling in bacteria. This assay will be a valuable tool as we explore the effect of cognate stimuli and protein-protein interactions on histidine kinase activity in various two-component signaling pathways.
The authors have nothing to disclose.
This work was supported by the Department of Education through the Graduate Assistance in Areas of National Need program (P200A100044).
Phosphoric acid | VWR | AAAA18067-AP | For quenching reactions and washing nitrocellulose |
Tris base | RPI | T60040-5000.0 | Tris-HCl pH 8.0, for kinase reaction buffer |
Potassium chloride | RPI | P41000-2500.0 | For kinase reaction buffer |
Magnesium chloride | RPI | M24000-500.0 | For kinase reaction buffer |
Glycerol | RPI | G22020-4000.0 | For kinase reaction buffer |
5'-ATP | Promega | E6011 | Kinase substrate |
[γ-32P]-5'-ATP | Perkin Elmer | NEG002Z250UC | 6000 Ci/mmol |
96-well dot blot apparatus | Bio-rad | 1706545 | For spotting reactions |
Nitrocellulose | Whatman | 32-10401396-PK | For spotting reactions |
Ponceau S | Sigma aldrich | P3504-50G | For staining nitrocellulose |