Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins

The overall goal of this methodology is to identify multiple phosphorylation of an intrinsically disordered protein by nuclear magnetic resonance spectroscopy using tau protein as a case study. This method can help to answer key question in molecular regulation related to disease such as the impact of the phosphorylation on the protein interaction with its molecular partner. The main advantage of this technique is that we can identify site specific phosphorylation in a disordered protein and link it with structural and functional changes.

This method can provide insight into the function. It can also be applied to other proteins such as the disordered remains of the DNA protein from the aPtc virus The applications of this technique extend toward therapy by development of protein, protein interaction inhibitors. This method will be demonstrated by Clement Danis, Clement Despres, Luiza Bessa, and Hamida Merzougui, the PhD students from our group.

To begin gently mix 50 microliters of competent BL21 cells with 100 nanograms of plasmid DNA forming 10 to 15 million colonies per microgram of plasma DNA in a 1.5 milliliter plastic tube. After placing the cell mixture on ice for 30 minutes, heat shock it for 10 seconds at 42 degrees celsius. Then place the tube back on ice for five minutes before adding one milliliter of room temperature Luria Bertani or LB medium.

Incubate the bacterial suspension at 37 degrees celsius for 30 minutes under gentle agitation. Using a spreader, spread 100 microliters of the cell suspension evenly onto an agar plate of LB medium containing 100 micrograms per milliliter of ampicillin antibiotic. Incubate the selection plate for 15 hours at 37 degrees celsius.

Next, solubilize 300 milligrams of Nitrogen 15 and Carbon 13 enriched medium, one gram of Nitrogen 15 Ammonium Chloride and two grams of Carbon 13 labelled glucose in 10 milliliters of M9 medium. Filter sterilize the isotope solution directly into the M9 medium using a 0.2 micron filter. Using a pasteur pipette suspend one colony of the recombinant plasma transformed bacteria from the selection plate in 20 milliliters of LB medium, supplemented with 100 micrograms per milliliter of ampicillin.

Incubate the inoculated medium at 37 degrees celsius for approximately six hours. Measure the optical density at 600 nanometers or OD600 using one milliliter of a ten-fold dilution of bacterial culture in a plastic spectrometer cuvette. Then, add 20 milliliters of the saturated LB culture to one liter of M9 growth medium supplemented with ampicillin in a two liter Erlenmeyer glass baffled culture flask.

Place the culture flask in a programmable incubator set to 10 degrees celsius and 50 RPM. Program the incubator to switch to 200 RPM and 37 degrees celsius early in the morning of the next day. Measure the OD600 on one milliliter of bacterial culture in a plastic spectrometer cuvette.

Add 400 microliters of one molar IPTG stock solution when the OD600 reaches a value of about 1.0 to induce the expression of recombinant tau protein. After incubating the bacterial cells at 37 degrees celsius for an additional three hours, collect them by centrifugation at 5, 000 times g for 20 minutes. Thaw the bacterial cell pellet and re-suspend it thoroughly in 45 milliliters of cation exchange buffer A, freshly supplemented with 1x protease inhibitor cocktail DNasel 1.

Disrupt the bacterial cells using a high-pressure homogenizer at 20, 000 PSI, three to four passes are necessary to adequately disrupt the cells. Centrifuge the disrupted cells at 20, 000 times g for 40 minutes to remove the insoluble material. Heat the bacterial cell extract for 15 minutes at 75 degrees celsius using a water bath.

A white precipitate will be observed after a few minutes. Then, centrifuge the heated extract at 15, 000 times g for 20 minutes and keep the supernatant containing the heat stable tau protein. Next, perform cation exchange chromatography on a strong CEX resin packed as a 5 milliliter bed column using a fast protein liquid chromatography system.

Set the flow rate at 2.5 milliliters per minute and equilibrate the column in CEX A buffer. Next, load the 60 to 70 milliliter heated tau extract using a sample pump, collect the flow-through for analysis to verify that the tau protein is efficiently binding to the resin. Wash the resin with CEX A buffer until the absorbance at 280 nanometers is back to the baseline value.

Program the FPLC with a gradient up to 25%CEX B buffer in 10 column volumes to reach 250 millimolar Sodium Chloride. Follow this with 50%CEX B buffer in five column volumes to reach 500 millimolar Sodium Chloride. Finally, increase the gradient to 100%CEX B buffer in two column volumes to reach one molar Sodium Chloride.

Collect 1.5 milliliter fractions during the elution steps. Then, perform a buffer exchange on the tau containing pooled fractions. Equilibrate a desalting column with a 53 milliliter G25 resin packed bed in 15 millimolar ammonium bicarbonate buffer using an FPLC system.

Set the flow rate to five milliliters per minute and inject the tau sample on the column via a five milliliter injection loop. Collect the fractions corresponding to the absorption peak at 280 nanometers. Pool all tau fractions before allocating the sample into the tubes.

Choose these tubes so that the volume of the solution is small compared to the volume of the tube. Next, punch holes in the tube caps using a needle before freezing the tau samples at minus 80 degrees celsius for later lyophilization. Solubilize four milligrams of lyophilized Nitrogen 15 and Carbon 13 enriched erk phosphorylated tau in 400 microliters of NMR buffer.

Add five percent deuterium oxide for field-locking of the NMR spectrometer and one millimolar TMSP as an internal NMR signal reference. Also, add 10 microliters of a 40 EK stock solution of a complete protease inhibitor cocktail. Transfer the sample in a five milliliter NMR tube using an electronic syringe with a long needle or using a pasteur pipette.

Close the NMR tube using the plunger. Remove any air bubbles trapped between the plunger and the liquid by moving the plunger. Place the NMR tube in a spinner.

Use the appropriate gauge to adjust its vertical position in the spinner such that most of the sample solution will be inside the NMR coil. Next, start the airflow in the NMR instrument by clicking lift in the Magnet Control System window. Carefully place the spinner with the tube in the airflow at the top of the magnet bore.

Then stop the airflow and let the tube descend into place inside the probe head within the magnet. After allowing the sample to equilibrate to 25 degrees celsius, type ATMM in the command line to perform semi-automatic tuning and matching of the probe head to optimize power transmission. Then click lock in the Magnet Control System window to engage the field frequency lock of the spectrometer.

Start the shimming procedure to optimize the homogeneity of the magnetic field at the position of the sample by first typing TopShim GUI on the command line to open the shim window. Then click start in the shim window. Check the residual B0 standard deviation to verify that the shims are optimal.

Next, calibrate the P1 parameter, which is the length of a proton radio frequency pulse in microseconds. This parameter is necessary to obtain a 90 degree rotation of proton spin magnetization. Aim for the 360 degree pulse using a one-dimensional spectrum of water protons.

Adjust the frequency offset by setting the O1 parameter to the proton water frequency in the one-dimensional spectrum. Finally, start the acquisition of one-dimensional proton spectrum by typing ZG in the command line. A representative result of purification of recombinant tau shows a major absorption peak at 280 nanometers observed during the elution gradient.

This peak corresponds to purified tau protein as seen on the acrylamide gel above the chromatogram. The chromatogram for desalting the recombinant purified tau shows that the absorption peak at 280 nanometers and the peak of conductivity is well separated ensuring that desalting of the protein is efficient for the lyophilization step. The one-dimensional NMR spectrum of recombinant nitrogen 15 tau at 900 megahertz confirms that an NMR signal from the protein sample can be detected.

Good signal to noise ratio indicates that the basic acquisition parameters were set correctly. The two-dimensional spectrum of the phosphorylated protein shows good resolution, resonances are narrow and well separated. The resonances in the region of the spectrum boxed in red correspond to the phosphorylated residues.

After watching this video you should be able to produce a recombinant protein with easy to pick labeling, to purify this protein and to set up the basic parameter for spectrum data acquisition. Once mastered, this protocol can be done in two weeks if it's organized properly. Don't forget that NMR spectroscopy uses very strong magnets that must not be approached with metallic objects.

While attempting this procedure it's important to remember that these other proteins are sensitive to protease lysis and a number of precautions have to be taken to avoid the degradation of the sample. Following this procedure, other methods like solid state NMR can be applied to investigate the amyloid-like aggregates that are often formed by disordered proteins. After its development, this technique paved the way for researchers in the field of chemical biology to explore active compounds with serapatic potential in vitro model systems.