Name: Nuclear Magnetic Resonance Spectroscopy to Identify Multiple Phosphorylations in Proteins
Uploaded: 2023-06-29T19:11:41
Description: Source: Danis, C. et. al., Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered ...

eoe sub articles

EoE Sub Articles

1. Production of 15N, 13C-Tau (Figure 1)
<ol>
	<li>ATransform pET15b-Tau recombinant T7 expression plasmid into BL21(DE3) competent Escherichia coli bacterial cells. 
	NOTE: the cDNA coding for the longest (441 amino acid residues) Tau isoform is cloned between NcoI and XhoI restriction sites in the pET15b plasmid.
	<ol>
		<li>Mix gently 50 &#181;l of competent BL21(DE3) cells, forming 1-5 x 107 colonies per &#181;g of plasmid DNA, with 100 ng of plasmid DNA in a 1.5 ml plastic tube. 
		NOTE: Codon-usage optimized bacterial strains for eukaryotic cDNA expression are not essential to produce human Tau.</li>
		<li>Place the cell mixture on ice for 30 min and then heat shock for 10 sec at 42 &#176;C. Place the tube back on the ice for 5 min and add 1 ml of room temperature LB (Luria-Bertani) medium. Incubate the bacterial suspension at 37 &#176;C for 30 min under gentle agitation.</li>
	</ol>
	</li>
	<li>Spread using an inoculation loop 100 &#181;l of cell suspension evenly onto an agar plate of LB medium containing 100 &#181;g/ml of ampicillin antibiotic.</li>
	<li>Incubate the selection plate for 15 hr at 37 &#176;C.</li>
	<li>Keep the selection plate at 4 &#176;C until proceeding to the culture step, for a maximum of 2 weeks approximately. 
	NOTE: a glycerol stock of bacterial culture (50% glycerol), stored at -80 &#176;C, can be prepared to start the culture at a later stage.</li>
	<li>Add 1 ml 1 M MgSO4, 1 ml 100 mM CaCl2, 10 ml 100x MEM vitamin complement, 1 ml 100 mg/ml ampicillin to 1 L of autoclaved M9 salts (6 g Na2HPO4, 3 g KH2PO4, 0.5 g NaCl). 
	NOTE: A white precipitate will form upon the addition of the CaCl2 solution to the M9 salts that quickly dissipates.</li>
	<li>Solubilize 300 mg of 15N, 13C-complete medium, 1 g of 15NH4Cl, and 2 g of 13C6-glucose in 10 ml of M9 medium. Filter-sterilize the isotope solution using a 0.2 &#181;m filter, directly into the M9 medium.</li>
	<li>Suspend using an inoculation loop one colony of pET15b-Tau transformed bacteria from the selection plate in 20 ml of LB medium supplemented with 100 &#181;g/ml of ampicillin.</li>
	<li>Incubate the inoculated medium at 37 &#176;C for about 6 hr.</li>
	<li>Measure optical density at 600 nm (OD600) on 1 ml of a ten-fold dilution of bacterial culture in a plastic spectrometer cuvette. 
	NOTE: Turbidity of the bacterial culture corresponding to OD600 of 3.0-4.0 indicates that the saturation growth phase is reached.</li>
	<li>Add 20 ml of the saturated LB culture to 1 L of M9 growth medium supplemented with ampicillin (100 &#181;g/ml final concentration), in a 2 L Erlenmeyer plastic baffled culture flask.</li>
	<li>Place the culture flask in a programmable incubator set to 10 &#176;C and 50 rpm. Program the incubator to switch to 200 RPM and 37 &#176;C early in the morning of the next day.</li>
	<li>Measure OD600 on 1 ml of bacterial culture in a plastic spectrometer cuvette. Add 400 &#181;l of 1 M IPTG (isopropyl &#946;-D-1-thiogalactopyranoside) stock solution (kept at -20 &#176;C) when OD600 reaches a value of about 1.0 to induce the expression of recombinant Tau protein.</li>
	<li>Continue the incubation at 37 &#176;C for a further 3 hr. Collect the bacterial cells by centrifugation at 5,000 x g for 20 min.</li>
	<li>Freeze the bacterial pellet at -20 &#176;C. Keep frozen until the purification step, for an extended period if needed.</li>
</ol>
2. Purification of 15N, 13C-Tau (Figure 2)
<ol>
	<li>Autoclave cation-exchange (CEX) purification buffers at 121 &#176;C under 15 psi for 20 min. Store buffers at 4 &#176;C.</li>
	<li>Thaw the bacterial cell pellet and resuspend thoroughly in 45 ml of extraction CEX A buffer (50 mM NaPi buffer pH 6.5, 1 mM EDTA) freshly supplemented with protease inhibitor cocktail 1x (1 tablet) and DNAseI (2,000 units).</li>
	<li>Disrupt the bacterial cells using a high-pressure homogenizer at 20,000 psi. 3-4 passes are necessary. Centrifuge at 20,000 x g for 40 min to remove insoluble material.</li>
	<li>Heat the bacterial cell extract for 15 min at 75 &#176;C using a water bath. 
	NOTE: a white precipitate is observed after a few minutes.</li>
	<li>Centrifuge at 15,000 x g for 20 min and keep the supernatant containing the heat-stable Tau protein.</li>
	<li>Store at -20 &#176;C until the following purification step if needed.</li>
	<li>Perform cation-exchange chromatography on a strong CEX resin packed as a 5 ml bed column using a fast protein liquid chromatography (FPLC) system (Figure 3 A).
	<ol>
		<li>Set flow rate to 2.5 ml/min.</li>
		<li>Equilibrate the column in CEX A buffer</li>
		<li>Load the 60-70 ml heated extract containing Tau using a sample pump, or alternatively pump A, depending on the system. Collect the flow-through for analysis to verify that Tau protein is efficiently binding to the resin (see 2.8).</li>
		<li>Wash the resin with CEX A buffer until the absorbance at 280 nm is back to the baseline value.</li>
		<li>Elute Tau from the column using a three-step NaCl gradient obtained by a gradual increase of CEX B buffer (CEX A buffer with 1 M NaCl). Program the FPLC as follows: the first step of the gradient to 25% CEX B buffer in 10 column volumes (CV) to reach 250 mM NaCl, the second step to 50% CEX B buffer in 5 CV to reach 500 mM NaCl, and the third step to 100% CEX B buffer in 2 CV to reach 1 M NaCl. Collect 1.5 ml fractions during the elution steps.</li>
	</ol>
	</li>
	<li>Analyze 10 &#181;l of the fractions collected during the elution step by SDS-PAGE (12% SDS-acrylamide gel) and Coomassie staining (Figure 3 A). Check the loading step on the column as well by analyzing 10 &#181;l of the flow-through.</li>
	<li>Choose the fractions containing Tau and pool these fractions for the next step.</li>
	<li>Perform a buffer exchange on Tau-containing pooled fractions (Figure 3 B).
	<ol>
		<li>Equilibrate a desalting column of 53 ml G25 resin-packed bed (26 x 10 cm) in 50 mM ammonium bicarbonate (volatile buffer) using an FPLC system.</li>
		<li>Set the flow rate to 5 ml/min. Inject the Tau sample on the column via a 5 ml injection loop. Collect fractions corresponding to the absorption peak at 280 nm.</li>
		<li>Repeat the injection 3-4 times, depending on the volume of the initial CEX pool.</li>
	</ol>
	</li>
	<li>Calculate the amount of purified Tau protein by using the peak area of the chromatogram at 280 nm (1 mg of Tau corresponds to 140 mAU*ml). 
	NOTE: The extinction coefficient of Tau protein at 280 nm is 7,550 M-1cm-1. Tau does not contain any Trp residues.</li>
	<li>Pool all Tau fractions.</li>
	<li>Aliquot the sample into tubes containing the equivalent of 1 to 5 mg of Tau. Choose these tubes so that the volume of solution is small compared to the volume of the tube (for example 5 ml of solution in a 50 ml tube).</li>
	<li>Punch holes in the tube caps using a needle. Freeze Tau samples at -80 &#176;C.</li>
	<li>Lyophilize Tau samples. Lyophilized Tau protein can be kept at -20 &#176;C for long periods of time.</li>
</ol>
3. In Vitro Phosphorylation of 15N-Tau
<ol>
	<li>Dissolve 5 mg of lyophilized Tau in 500 &#181;l phosphorylation buffer (50 mM Hepes&#183;KOH, pH 8.0, 12.5 mM MgCl2, 1 mM EDTA, 50 mM NaCl).</li>
	<li>Add 2.5 mM ATP (25 &#181;l of 100 mM stock solution kept at -20 &#176;C), 1 mM DTT (1 &#181;l of 1 M stock solution kept at -20 &#176;C), 1 mM EGTA (2 &#181;l of a 0.5 M stock solution), 1x protease inhibitor cocktail (25 &#181;l of a 40x stock obtained by dissolving 1 tablet in 1 ml phosphorylation buffer) and 1 &#181;M activated His-ERK2 (250 &#181;l in conservation buffer 10 mM Hepes, pH 7.3, 1 mM DTT, 5 mM MgCl2, 100 mM NaCl and 10% glycerol, stored at -80 &#176;C) in a total sample volume of 1 ml. 
	NOTE: The activated His-ERK2 can be prepared in-house by phosphorylation with the MEK kinase.</li>
	<li>Incubate 3 hr at 37 &#176;C.</li>
	<li>Heat the sample at 75 &#176;C for 15 min to inactivate ERK kinase.</li>
	<li>Centrifuge at 20,000 x g for 15 min. Collect and keep the supernatant.</li>
	<li>Desalt the protein sample into 50 mM ammonium bicarbonate using a column of 3.45 ml G25 resin-packed bed (1.3 x 2.6 cm), which is suitable for a 1 ml sample.</li>
	<li>Run a 12% SDS-PAGE with 2.5 &#181;l of the protein sample to check both its integrity and efficient phosphorylation (Figure 4).</li>
	<li>Lyophilize the phosphorylated Tau sample. Store the powder at -20 &#176;C.</li>
</ol>
4. Acquisition of NMR Spectra (Figure 5)
<ol>
	<li>Solubilize 4 mg of lyophilized 15N, 13C ERK-phosphorylated-Tau in 400 &#181;l NMR buffer (50 mM NaPi or 50 mM deuterated Tris-d11.Cl, pH 6.5, 30 mM NaCl, 2.5 mM EDTA, and 1 mM DTT).</li>
	<li>Add 5% D2O for field locking of the NMR spectrometer and 1 mM TMSP (3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt)) as internal NMR signal reference. Add 10 &#181;l of a 40x stock solution of a complete protease inhibitor cocktail.</li>
	<li>Transfer the sample to a 5 mm NMR tube using an electronic syringe with a long needle or Pasteur pipette. Close the NMR tube using the plunger. Remove any air bubbles trapped between the plunger and the liquid by plunger movements.</li>
	<li>Place the NMR tube in a spinner. Adjust its vertical position in the spinner with the appropriate gauge for the NMR probe head used, such that most of the sample solution will be inside the NMR coil.</li>
	<li>Start the airflow: click lift in the magnet control system window. Carefully place the spinner with the tube in the airflow at the top of the magnet bore. Stop the airflow (click lift) and let the tube descend into place inside the probe head in the magnet.</li>
	<li>Set the temperature to 25 &#176;C (298 K).</li>
	<li>Perform semi-automatic tuning and matching of the probe head to optimize power transmission. Type atmm on the command line.</li>
	<li>Lock the spectrometer frequency using the D2O signal of the sample recorded on the deuterium channel. Click lock in the magnet control system window.</li>
	<li>Start the shimming procedure to optimize the homogeneity of the magnetic field at the position of the sample. Type topshim gui on the command line to open the shim window. Click Start in the shim window. Check the residual B0 standard variation value to verify that shims are optimal (less than 2 Hz is good).</li>
	<li>Calibrate the p1 parameter (length of a proton radiofrequency pulse in &#181;sec), which is necessary to obtain a 90&#176; rotation of proton spins. Aim for the 360&#176; pulse using a 1D spectrum of water protons (Figure 6).</li>
	<li>Adjust the frequency offset by setting the o1 parameter (in Hz) to the proton water frequency in the 1D spectrum (Figure 6).</li>
	<li>Start acquisition of a 1D proton spectrum (pulse sequence with watergate sequence for water signal suppression, for example, zggpw5) to verify signals from the sample (Figure 7). Adapt the number of scans to the relative protein concentration. Type zg on the command line to start the acquisition.</li>
</ol>

No conflicts of interest declared.

nuclear magnetic resonance spectroscopy to identify multiple phosphorylations in proteins

Source: Danis, C. et. al., <a href="https://app.jove.com/v/55001">Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins.</a>&#160;J. Vis. Exp.&#160; (2016).
This video demonstrates the use of nuclear magnetic resonance spectroscopy (NMR) techniques to identify multiple phosphorylations in a protein. The phosphorylation of a protein at specific amino acid causes the deshielding of the neighboring amide hydrogen, which generates the spectral difference.

<img alt="Figure 1" src="/files/ftp_upload/21467/21467_Figure1.jpg" /> 
Figure 1: Scheme of the main steps of recombinant protein production and isotopic labeling.&#160;Steps from bacteria transformation to recombinant protein production are outlined as described in paragraph 1 of the protocol.
<img alt="Figure 2" src="/files/ftp_upload/21467/21467_Figure2.jpg" /> 
Figure 2: Scheme of the main ...

Nuclear Magnetic Resonance Spectroscopy to Identify Multiple Phosphorylations in Proteins

Source: Danis, C. et. al., Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered ...

Multiple phosphorylations in a protein involve adding phosphate groups to protein at various positions, specifically at serine and threonine residues.
To identify multiple phosphorylations in a protein, begin with a tube containing phosphorylated proteins labeled with nitrogen-15, which helps to differentiate amide hydrogens from other hydrogens in the protein.
Add an internal NMR reference compound for accurate measurement of the relative resonance frequencies. Transfer the mixture to a nuclear magnetic resonance, or NMR, tube. Insert the tube into a spinning aperture, and place it in an NMR spectrometer.
The presence of phosphates exhibits electron-withdrawing effects in the phosphorylated serine and threonine residues. As a result, the amide hydrogens of phosphorylated residues appear to be deshielded with lesser electron densities compared to the other amide hydrogens.
Amide hydrogens with varying electronic environments experience differential magnetic fields around nuclei, attaining low-energy states.
Apply a brief radiofrequency pulse to excite the hydrogen nuclei to a higher energy state at a specific resonance frequency based on their electronic environment.
During relaxation, the nuclei return to a low-energy state by releasing the absorbed energy, which is detected to produce an NMR signal.
Plot a two-dimensional NMR spectrum representing the individual amide hydrogens.
Compare the spectrum of the phosphorylated protein with that of the non-phosphorylated protein. The appearance of additional resonances in the phosphorylated protein&#8217;s NMR spectrum confirms multiple phosphorylations.

nuclear-magnetic-resonance-spectroscopy-to-identify-multiple

Research

JoVE Encyclopedia of Experiments

Biological Techniques

Spectroscopy Techniques

194 Views. Source: Danis, C. et. al., Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins. J. Vis. Exp.  (2016). This video demonstrates the use of nuclear magnetic resonance spectroscopy (NMR) techniques to identify multiple phosphorylations in a protein. The phosphorylation of a protein at specific amino acid causes the deshielding of the neighboring amide hydrogen, which generates the spectral difference. 

Video: Nuclear Magnetic Resonance Spectroscopy to Identify Multiple Phosphorylations in Proteins - Concept

NMR Spectroscopy as a Robust Tool for the Rapid Evaluation of the Lipid Profile of Fish Oil Supplements

The western diet is poor in n-3 fatty acids, therefore the consumption of fish oil supplements is recommended to increase the intake of these essential nutrients. The objective of this work is to demonstrate the qualitative and quantitative analysis of encapsulated fish oil supplements using high-resolution 1H and 13C NMR spectroscopy utilizing two different NMR instruments; a 500 MHz and an 850 MHz instrument. Both proton (1H) and carbon (13C) NMR spectra can be used for the quantitative determination of the major constituents of fish oil supplements. Quantification of the lipids in fish oil supplements is achieved through integration of the appropriate NMR signals in the relevant 1D spectra. Results obtained by 1H and 13C NMR are in good agreement with each other, despite the difference in resolution and sensitivity between the two nuclei and the two instruments. 1H NMR offers a more rapid analysis compared to 13C NMR, as the spectrum can be recorded in less than 1 min, in contrast to 13C NMR analysis, which lasts from 10 min to one hour. The 13C NMR spectrum, however, is much more informative. It can provide quantitative data for a greater number of individual fatty acids and can be used for determining the positional distribution of fatty acids on the glycerol backbone. Both nuclei can provide quantitative information in just one experiment without the need of purification or separation steps. The strength of the magnetic field mostly affects the 1H NMR spectra due to its lower resolution with respect to 13C NMR, however, even lower cost NMR instruments can be efficiently applied as a standard method by the food industry and quality control laboratories.

Here, high-resolution 1H and 13C Nuclear Magnetic Resonance (NMR) spectroscopy was used as a rapid and reliable tool for quantitative and qualitative analysis of encapsulated fish oil supplements.

The western diet is poor in n-3 fatty acids, therefore the consumption of fish oil supplements is recommended to increase the intake of these essential ...

JoVE Journal

Chemistry

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment screening. NMR-based activity assays are ideally suited to work at the higher concentrations of test compounds required to detect these weaker inhibitors. The dynamic range and chemical shift dispersion in an NMR experiment can easily resolve resonances from substrate, product, and test compounds. This contrasts with spectrophotometric assays, in which read-out interference problems often arise from compounds with overlapping UV-vis absorption profiles. In addition, since they lack reporter enzymes, the single-enzyme NMR assays are not prone to coupled-assay false positives. This attribute makes them useful as orthogonal assays, complementing traditional high throughput screening assays and benchtop triage assays. Detailed protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells. The methods are demonstrated using two nucleoside ribohydrolase enzymes. The use of 1H NMR is shown for the purine-specific enzyme, while 19F NMR is shown for the pyrimidine-specific enzyme. The protocols are generally applicable to any enzyme where substrate and product resonances can be observed and distinguished by NMR spectroscopy. To be the most useful in the context of drug discovery, the final concentration of substrate should be no more than 2-3x its Km value. The choice of NMR experiment depends on the enzyme reaction and substrates available as well as available NMR instrumentation.

NMR-Based Activity Assays for Determining Compound Inhibition, IC50 Values, Artifactual Activity, and Whole-Cell Activity of Nucleoside Ribohydrolases

NMR-based activity assays have been developed to identify and characterize inhibitors of two nucleoside ribohydrolase enzymes. Protocols are provided for initial compound assays at 500 &#956;M and 250 &#956;M, dose-response assays for determining IC50 values, detergent counter screen assays, jump-dilution counter screen assays, and assays in E. coli whole cells.

NMR spectroscopy is often used for the identification and characterization of enzyme inhibitors in drug discovery, particularly in the context of fragment ...

Isolation of Whole Cell Protein Lysates from Mouse Facial Processes and Cultured Palatal Mesenchyme Cells for Phosphoprotein Analysis

Mammalian craniofacial development is a complex morphological process during which multiple cell populations coordinate to generate the frontonasal skeleton. These morphological changes are initiated and sustained through diverse signaling interactions, which often include protein phosphorylation by kinases. Here, two examples of physiologically-relevant contexts in which to study phosphorylation of proteins during mammalian craniofacial development are provided: mouse facial processes, in particular E11.5 maxillary processes, and cultured mouse embryonic palatal mesenchyme cells derived from E13.5 secondary palatal shelves. To overcome the common barrier of dephosphorylation during protein isolation, adaptations and modifications to standard laboratory methods that allow for isolation of phosphoproteins are discussed. Additionally, best practices are provided for proper analysis and quantification of phosphoproteins following western blotting of whole cell protein lysates. These techniques, particularly in combination with pharmacological inhibitors and/or murine genetic models, can be used to gain greater insight into the dynamics and roles of various phosphoproteins active during craniofacial development.

The protocol presents a method for isolating whole cell protein lysates from dissected mouse embryo facial processes or cultured mouse embryonic palatal mesenchyme cells and performing subsequent western blotting to assess phosphorylated protein levels.

Mammalian craniofacial development is a complex morphological process during which multiple cell populations coordinate to generate the frontonasal ...

Nuclear Magnetic Resonance Spectroscopy to Identify Multiple Phosphorylations in Proteins

Transcript