The NMR facilities were funded by the R&#233;gion Nord, CNRS, Pasteur Institute of Lille, European Community (FEDER), French Research Ministry and the University of Sciences and Technologies of Lille. We acknowledge support from the TGE RMN THC (FR-3050, France), FRABio (FR 3688, France) and Lille NMR and RPE Health and Biology core facility. Our research is supported by grants from the LabEx (Laboratory of Excellence) DISTALZ (Development of Innovative Strategies for a Transdisciplinary approach to Alzheimer's disease), EU ITN TASPPI and ANR BinAlz.

1. Production of 15N, 13C-Tau (Figure 1)
<ol>
	<li>Transform pET15b-Tau recombinant T7 expression plasmid13,14 into BL21(DE3) competent Escherichia coli bacterial cells15. 
	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 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 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 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 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 15 N, 13 C-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 a 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 absorbance at 280 nm is back to baseline value.</li>
		<li>Elute Tau from the column using a three-step NaCl gradient obtained by gradual increase of CEX B buffer (CEX A buffer with 1 M NaCl). Program the FPLC as follows: first step of the gradient to 25% CEX B buffer in 10 column volumes (CV) to reach 250 mM NaCl, second step to 50% CEX B buffer in 5 CV to reach 500 mM NaCl, and 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)16. 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 a FPLC system.</li>
		<li>Set 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-house5,8 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-PAGE16 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 complete protease inhibitor cocktail.</li>
	<li>Transfer the sample in 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 bubble trapped between plunger and 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 air flow: 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 air flow (click lift) and let the tube descend into place inside the probe head in the magnet.</li>
	<li>Set 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 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>
	<li>Set up additional parameters for the acquisition of a 2D [1H,15N] HSQC spectrum (pulse sequence hsqcetfpf3gpsi, Figures 8-9).
	<ol>
		<li>For a 15N,13C labeled sample, decouple 13C during the 15N indirect evolution.</li>
		<li>Set the number of points and the spectral width (ppm) in the 1H (F2) and 15N (F1) dimensions. 
		NOTE: Adapt the number of acquisition data points to the spectrometer field to keep a similar number of Hz per point and to limit decoupling times: use 3,072 points at 900 MHz and 2,048 points at 600 MHz, in the 1H dimension.</li>
		<li>Optimize additional parameters in the pulse sequences, corresponding to delays, pulse lengths, offset frequencies, power levels. Type ased on the command line to display all parameters relevant for the experiment.</li>
	</ol>
	</li>
	<li>Set parameters for the acquisition of a 3D [1H,15N,13C] HNCACB spectrum (pulse sequence hncacbgpwg3d, Figure 10A) at 600 MHz.</li>
	<li>Set parameters for a 3D [1H,15N,15N] HNCANNH experiment (pulse sequence hncannhgpwg3d) at 600 MHz. Set the number of points to 2048 in the 1H and, 64 and 128 points in the two 15N dimensions. Define the spectral widths as 14, 25, 25 parts per million (ppm) centered on 4.7, 119, 119 ppm in the 1H, 15N and 15N dimensions. Duration of acquisition with 16 scans is 1 day and 22 hr.</li>
</ol>
5. Identification of Phosphorylation Sites
<ol>
	<li>Process spectra using acquisition and processing NMR software.
	<ol>
		<li>Perform a Fourier transformation of the data (Figure 7). Type ft for a 1D spectrum, xfb for a 2D spectrum or ft3d for a 3D spectrum, on the command line.</li>
		<li>Phase and reference all spectra (Figure 7C) using the interactive windows.</li>
	</ol>
	</li>
	<li>Identify resonances of interest in the 2D HSQC potentially corresponding to phosphorylated Ser and Thr residues (Figure 9, red box).</li>
	<li>Extract planes (i.e. 2D 1H-13C spectra) from the 3D 1H-15N-13C spectrum using the 15N chemical shifts of resonances of interest in the 2D. Use the cursor of dimension 2 (w2) to choose the 15N frequency corresponding to the plane (w1-w3) to be visualized (Figure 10B).</li>
	<li>Pick the resonance frequencies of &#39;CA&#39; and &#39;CB&#39; 13C nuclei of the &#39;i&#39; and &#39;i-1&#39; residues (weaker set of signals compared to those of the i residue) for each [1H, 15N] resonance of interest in the HNCACB 3D spectrum by clicking on the resonance, in pointer mode menu find/add peak, to add the chemical shift value in a peak list file.
	<ol>
		<li>Identify the i residue type, pSer or pThr, by comparing the chemical shifts in the peak list to known values of CA and CB chemical shifts of pSer and pThr17.</li>
		<li>Identify the presence of a Pro residue at the i+1 position by a characteristic additional +2 ppm shift of the CA chemical shift value18.</li>
		<li>Compare the chemical shift values of the CA and CB resonances corresponding to the i-1 residue to a table of chemical shifts predicted for Tau amino acid residues19 to identify the nature of the residue at the i-1 position.</li>
	</ol>
	</li>
	<li>Pick the resonance frequencies of 15N nuclei of the &#39;i&#39; and &#39;i-1&#39; residues for each [1H, 15N] resonance of interest in the HNCANNH spectrum.</li>
	<li>Compare the 15N chemical shift values to the chemical shift assignment of the Tau protein20-23.</li>
	<li>Compare the identified dipeptides with the Tau sequence to define the sequence specific assignment.</li>
</ol>

The authors have nothing to disclose.

We have used NMR spectroscopy to characterize enzymatically modified Tau samples. The recombinant expression and purification described here for the full-length human Tau protein can similarly be used to produce mutant Tau or Tau domains. Isotopically enriched protein is needed for NMR spectroscopy, necessitating recombinant expression. Identification of phosphorylation sites requires resonance assignment and a 15N, 13C doubly labeled protein. Given the cost of isotopes, good yield is required in the recombinant expression step. Glucose is the limiting factor for the bacterial growth in the M9 medium therefore the amount of 13C6-glucose can be increased to 4 g per liter of growth medium to improve yield. Addition of complete medium and MEM vitamins are not compulsory but help to stimulate growth and improve yield. Given the high cost of the complete labeled medium, this product is only used as a growth medium supplement. Bacterial growth is slow in M9 medium. Generally, bacterial cultures using M9 media supplemented with 3% complete medium reach time of induction after about 4 hr of incubation. An OD600 of 1.6-1.8 is usually obtained at the end of the culture. Expected yield of recombinant Tau protein is about 15 mg per liter of bacterial culture. The use of a programmable incubator allows to conveniently schedule protein production, collection of the bacterial pellet and analytical control of protein production during working day hours.
Sample concentration is important to obtain a good quality spectrum. A typical Tau sample sufficient for a 2D spectrum would contain 1 mg in 200 &#181;l (i.e. 100 &#181;M) Tau protein. For a 3D spectrum, at least 200 &#181;M in 300 &#181;l are required, provided that a cryogenic probe head is used. Access to a high-field spectrometer, such as the 900 MHz instrument used in this study will provide better signal-to-noise and reduce constraints on sample concentration (Figure 8). Given that Tau is a large disordered protein, its NMR spectra are characterized by considerable signal overlap, and a high-field NMR spectrometer will also be the best choice in terms of resolution (Figure 8). Nevertheless, the resonances corresponding to phosphorylation sites are in a distinct region of the spectrum and are easy to detect even with a 600 MHz spectrometer (Compare Figures 9B and 9C). In addition, due to its disordered nature, the Tau protein is sensitive to proteolysis (Figures 8B).
Sterilization of buffers is advised to limit Tau degradation. Addition of protease inhibitors to the NMR sample helps to protect Tau against degradation during data acquisition periods that can last from hours to days, depending on the pulse sequence. Low pH (i.e. pH below 7.0) is required to avoid too fast exchange between protein protons and water protons, which leads to signal broadening. Sample pH must also be well controlled to ensure reproducibility of the spectra. Indeed, since pKa values of pSer and pThr are close to the optimal pH for NMR spectroscopy, chemical shifts of phosphorylated residues are very sensitive to pH variations. Adjustment of the pH of the NMR sample can be made directly using a pH-meter with a pH micro electrode, adapted to small volumes. Tau is a soluble protein and does not aggregate in standard conditions. Addition of polyanions, such as heparin sulfate, and incubation at 37 &#176;C can initiate aggregation in Tau samples. In this case, given the solid nature of the aggregates, most of the resonances in the corresponding NMR spectrum broaden beyond detection. Even the phosphorylated Tau samples do not aggregate in the conditions described here for NMR data acquisition.
Compared to antibody analysis, NMR provides an overall view of PTMs. Mass spectrometry can also be used to identify the phosphorylation pattern of a protein sample. Isotopic labeling is not necessary for this technique, and required sample quantities are much smaller. However, characterization of a protein with multiple phosphorylations, such as Tau, remains challenging. Adjacent phosphorylations will produce isobaric peptides in bottom-up strategies of identification. Complete identification then needs MS/MS sequencing of the peptides obtained by sample proteolysis. An advantage of NMR consists in the intrinsic quantitative nature of the technique. The intensity of an NMR signal can be linked to the amount of the chemical group present at a specific site. We can thus define the proportion of chemical modification at each site. Most recent progress in MS applications have however shown that MS analysis of Tau multiple phosphorylation is feasible24, even in a quantitative manner after proper normalization25.
Here, we presented Tau phosphorylation by activated ERK2, but the methodology can be used for phosphorylation with other kinases as well6,7,26-28. Kinetic experiments can be performed, which can help to define kinase specificity towards a protein substrate28-31. Phosphorylation studies are not limited to recombinant kinases, and kinase activity of cell or tissue extracts can be analysed6,32,28. An interesting development is the use of in-cell NMR to study in situ modifications33,34. Conversely, NMR is also well-adapted to address phosphatase specificity, as was shown by studying the dephosphorylation of phosphorylated Tau by the PP2A phosphatase35. NMR spectroscopy can be applied to the characterization of kinase inhibitors by comparing the phosphorylation profile of the protein substrate in a 2D spectrum in presence of the inhibitor compound with the spectrum issued from a control experiment6.
The interest of using NMR spectroscopy, compared to the more sensitive MS techniques, resides in the wide range of applications exploiting the protocol described here, rather than on its analytical capacity alone. It has proven crucial to identify phosphorylation sites to be able to link specific phosphorylations with structural or functional modifications that were mainly studied using NMR. NMR spectroscopy of phosphorylated Tau samples allows to explore the structural impact of phosphorylation on both transient local secondary structures and on global rearrangement of the dynamic ensemble of modified Tau36,37. Functional aspects include the regulation of both interaction of Tau with protein partners7,38,39 and aggregation by Tau phosphorylation8. The phosphorylated Tau sample characterized by NMR can be further used to decipher phospho-dependent interactions, for example with 14-3-3 proteins40, and engineered protein-protein interaction inhibitors41,42. NMR allows definition of interaction site(s) at the residue level, and of the dependence of this interaction on phosphorylation. Additionally, NMR spectroscopy of phosphorylated Tau is a key methodology to characterize the proline cis:trans isomerase Pin1, an important phospho-dependent enzyme involved in Tau regulation43-45. In addition, phosphorylation analysis by NMR can be applied not only to IDPs but also to globular proteins46. Finally, other types of Tau PTMs such as acetylations22,40,41 can be studied by NMR. The protocols described here have proven crucial to better understand the functional and structural regulation of Tau in physiological and pathological conditions.

One of the main challenges of healthcare in the 21st century are neurodegenerative diseases such as Alzheimer&#180;s disease (AD). Tau is a microtubule-associated protein that stimulates microtubule (MT) formation. Tau is equally involved in several neurodegenerative disorders, so-called tauopathies, of which the best known is AD. In these disorders, Tau self-aggregates in paired helical filaments (PHFs) and is found modified on many residues by posttranslational modifications (PTMs) such as phosphorylation1. Phosphorylation of Tau protein is implicated in both regulation of its physiological function of MT stabilization and pathological loss of function that characterizes AD neurons.
Furthermore, Tau protein, when integrated in PHFs in diseased neurons, is invariably hyperphosphorylated2. Unlike normal Tau that contains 2-3 phosphate groups, the hyperphosphorylated Tau in PHFs contains 5 to 9 phosphate groups3. Hyperphosphorylation of Tau corresponds both to an increase of stoichiometry at some sites and to phosphorylation of additional sites that are called pathological sites of phosphorylation. However, overlap exists between AD and normal adult patterns of phosphorylation, despite quantitative differences in the level4. How specific phosphorylation events influence function and dysfunction of Tau remains largely unknown. We aim to decipher Tau regulation by PTMs at the molecular level.
To deepen the understanding of the molecular aspects of Tau, we have to address technical challenges. Firstly, Tau is an intrinsically disordered protein (IDP) when isolated in solution. Such proteins lack well-defined three-dimensional structure under physiological conditions and require particular biophysical methods to study their function(s) and structural properties. Tau is a paradigm for the growing class of IDPs, often found associated with pathologies such as neurodegenerative diseases, hence increasing the interest to understand the molecular parameters underlying their functions. Secondly, characterization of Tau phosphorylation is an analytical challenge, with 80 potential phosphorylation sites along the sequence of the longest 441 amino-acid Tau isoform. A number of antibodies have been developed against phosphorylated epitopes of Tau and are used for detection of pathological Tau in neurons or brain tissue. Phosphorylation events can take place on at least 20 sites targeted by proline-directed kinases, most of them in close proximity within the Proline-rich region. The qualitative (which sites?) and quantitative (what stoichiometry?) characterization is difficult even by the most recent MS techniques5.
NMR spectroscopy can be used to investigate disordered proteins that are highly dynamic systems constituted of ensembles of conformers. High-resolution NMR spectroscopy was applied to investigate both structure and function of the Tau protein. In addition, the complexity of Tau&#39;s phosphorylation profile led to the development of molecular tools and new analytical methods using NMR for the identification of phosphorylation sites6-8. NMR as an analytical method allows for the identification of Tau phosphorylation sites in a global manner, visualization of all the single-site modifications in a single experiment, and quantification of the extent of phosphate incorporation. This point is essential since although phosphorylation studies on Tau abound in the literature, most of them have been performed with antibodies, leaving a large degree of uncertainty over the complete profile of phosphorylation and thus the true impact of individual phosphorylation events. Recombinant kinases including PKA, Glycogen-synthase kinase 3&#946; (Gsk3&#946;), Cyclin-dependent kinase 2/cyclin A (CDK2/CycA), Cyclin-dependent kinase 5 (CDK5)/p25 activator protein, extracellular-signal-regulated kinase 2 (ERK2) and microtubule-affinity-regulating kinase (MARK), which show phosphorylation activity towards Tau, can be prepared in an active form. In addition, Tau mutants that allow for generating specific Tau protein isoforms with well-characterized phosphorylation patterns are used to decipher the phosphorylation code of Tau. NMR spectroscopy is then used to characterize enzymatically modified Tau samples6-8. Although in vitro phosphorylation of Tau is more challenging than pseudo-phosphorylation such as by mutation of selected Ser/Thr into glutamic acid (Glu) residues, this approach has its merits. Indeed, neither the structural impact nor interaction parameters of phosphorylation can always be mimicked by glutamic acids. An example is the turn motif observed around phosphoserine 202 (pSer202)/phosphothreonine 205 (pThr205), which is not reproduced with Glu mutations9.
Here, the preparation of isotopically labeled Tau for NMR investigations will be described first. Tau protein phosphorylated by ERK2 is modified on numerous sites described as pathological sites of phosphorylation, and thus represents an interesting model of hyperphosphorylated Tau. A detailed protocol of Tau in vitro phosphorylation by recombinant ERK2 kinase is presented. ERK2 is activated by phosphorylation by mitogen activated protein kinase/ERK kinase (MEK)10-12. In addition to the preparation of modified, isotopically-labeled Tau protein, the NMR strategy used for identification of the PTMs is described.

<table><tbody><tr><td>pET15B recombinant T7 expression plasmid</td><td>Novagen</td><td>69257</td><td>Keep at -20 &amp;deg;C</td></tr><tr><td>BL21(DE3) transformation competent &lt;em&gt;E. coli&lt;/em&gt; bacteria</td><td>New England Biolabs</td><td>C2527I</td><td>Keep at -80 &amp;deg;C</td></tr><tr><td>Autoclaved LB Broth, Lennox&amp;nbsp;</td><td>DIFCO</td><td>240210</td><td>Bacterial Growth Medium</td></tr><tr><td>MEM vitamin complements 100x</td><td>Sigma</td><td>58970C</td><td>Bacterial Growth Medium Supplement</td></tr><tr><td>&lt;sup&gt;15&lt;/sup&gt;N, &lt;sup&gt;13&lt;/sup&gt;C-ISOGRO complete medium powder</td><td>Sigma</td><td>608297</td><td>Bacterial Growth Medium Supplement</td></tr><tr><td>&lt;sup&gt;15&lt;/sup&gt;NH&lt;sub&gt;4&lt;/sub&gt;Cl</td><td>Sigma</td><td>299251</td><td>Isotope</td></tr><tr><td>&lt;sup&gt;13&lt;/sup&gt;C&lt;sub&gt;6&lt;/sub&gt;-Glucose</td><td>Sigma</td><td>389374</td><td>Isotope</td></tr><tr><td>Protease inhibitor tablets&amp;nbsp;</td><td>Roche</td><td>5056489001</td><td>Keep at 4 &amp;deg;C</td></tr><tr><td>1 tablet in 1 ml is 40x solution that can be kept at -20 &amp;deg;C</td><td></td><td></td><td></td></tr><tr><td>DNaseI</td><td>EUROMEDEX</td><td>1307</td><td>Keep at -20 &amp;deg;C</td></tr><tr><td>Homogenizer (EmulsiFlex-C3)</td><td>AVESTIN</td><td></td><td>Lysis is realized at 4 &amp;deg;C</td></tr><tr><td>Pierce&amp;trade; Unstained Protein MW Marker</td><td>Pierce</td><td>266109</td><td></td></tr><tr><td>Active human MEK1 kinase, GST Tagged</td><td>Sigma</td><td>M8822</td><td>Keep at -80 &amp;deg;C</td></tr><tr><td>AKT&amp;Auml; Pure chromatography system</td><td>GE Healthcare</td><td></td><td>FPLC</td></tr><tr><td>HiTrap SP Sepharose FF (5 ml column)</td><td>GE Healthcare</td><td>17-5156-01</td><td>Cation exchange chromatography columns</td></tr><tr><td>HiPrep 26/10 Desalting</td><td>GE Healthcare</td><td>17-5087-01</td><td>Protein Desalting column</td></tr><tr><td>PD MidiTrap G-25</td><td>GE Healthcare</td><td>28-9180-08</td><td>Protein Desalting column</td></tr><tr><td>Tris D11, 97% D</td><td>Cortecnet</td><td>CD4035P5</td><td>Deuterated NMR buffer</td></tr><tr><td>5 mm Symmetrical Microtube SHIGEMI D&lt;sub&gt;2&lt;/sub&gt;O (set of 5 inner &amp;amp; outerpipe)&amp;nbsp;</td><td>Euriso-top</td><td>BMS-005B</td><td>NMR Shigemi Tubes</td></tr><tr><td>eVol kit-electronic syringe starter kit</td><td>Cortecnet</td><td>2910000</td><td>Pipetting</td></tr><tr><td>Bruker 900 MHz AvanceIII with a triple resonance cryogenic probehead</td><td>Bruker</td><td></td><td>NMR spectrometer for data acquisition</td></tr><tr><td>Bruker 600 MHz Avance with a triple resonance cryogenic probehead</td><td>Bruker</td><td></td><td>NMR spectrometer for data acquisition</td></tr><tr><td>TopSpin 3.1</td><td>Bruker</td><td></td><td>Acquisition and Processing software for NMR experiments</td></tr><tr><td>Sparky 3.114</td><td>UCSF (T. D. Goddard and D. G. Kneller)</td><td></td><td>NMR data Analysis software</td></tr></tbody></table>

nuclear magnetic resonance spectroscopy for the identification of multiple phosphorylations of intrinsically disordered proteins

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, abnormal phosphorylation of Tau. In the course of the molecular investigation of Tau functions and dysfunctions in the disease, nuclear magnetic resonance (NMR) spectroscopy is used to identify the multiple phosphorylations of Tau. We present here detailed protocols of recombinant production of Tau in bacteria, with isotopic enrichment for NMR studies. Purification steps that take advantage of Tau's heat stability and high isoelectric point are described. The protocol for in vitro phosphorylation of Tau by recombinant activated ERK2 allows for generating multiple phosphorylations. The protein sample is ready for data acquisition at the issue of these steps. The parameter setup to start recording on the spectrometer is considered next. Finally, the strategy to identify phosphorylation sites of modified Tau, based on NMR data, is explained. The benefit of this methodology compared to other techniques used to identify phosphorylation sites, such as immuno-detection or mass spectrometry (MS), is discussed.

Figure 3A shows a major absorption peak at 280 nm observed during the elution gradient. This peak corresponds to purified Tau protein as seen on the acrylamide gel above the chromatogram. Figure 3B shows a well separated absorption peak at 280 nm and peak of conductivity, ensuring that desalting of the protein is efficient. Figure 4 shows protein gel-shift observed by SDS-PAGE analysis16 characteristic of multiple protein phosphorylation (compare lanes 2 and 3). Figure 6 shows a series of proton (1H) 1D spectra with increasing pulse lengths (in &#181;sec). To setup the length of the pulse that will rotate 1H spin magnetization by 90&#176;, a pulse corresponding to a 360&#176; rotation is used in practice, as it is easier to calibrate by minimizing the signal. The signal of water protons is null when the 360&#176; pulse length is adequately defined. The value corresponding to a 360&#176; rotation is then divided by 4. p1 in this experiment is 10.5 &#181;sec. Enlarged region: The frequency of the residual signal is used to define the o1p parameter (frequency offset for 1H). Figure 7A. shows a free induction decay (FID), visualized to ensure that an NMR signal is detected. Figure 7B. shows a 1D 1H spectrum with an incorrect phase, as seen by resonances appearing as asymmetric peaks. Figure 7C shows a 1D 1H spectrum with good signal to noise ratio, indicating that the basic acquisition parameters were correctly set and a signal from the protein sample can be detected. Figure 8 shows 2D 1H,15N HSQC spectra of recombinant 15N-Tau at 900 MHz; Figure 8A with good sensitivity and resolution and Figure 8B. with detection of proteolysis in the sample, as seen by the appearance of additional peaks in the spectrum (blue box). Figure 9 shows 2D 1H,15N HSQC spectra of recombinant 15N-Tau Figure 9A at 600 MHz, with good sensitivity but less resolution compared to Figure 8A. Figure 9B at 600 MHz, shows appearance of additional peaks in the spectrum corresponding to phosphorylated residues (red box). Figure 9C at 900 MHz, shows peaks in the spectrum corresponding to phosphorylated residues (red box).&#160;Resolution is better than in Figure 9B. Figure 10A shows projections from the 3D NMR spectrum used to evaluate a successful experiment. Figure 10B shows a 1H-13C plane extracted from the 1H-15N-13C 3D spectrum with good signal intensity allowing to detect 13C signals from both i and i-1 residues.
<img alt="Figure 1" src="/files/ftp_upload/55001/55001fig1.jpg" /> 
Figure 1: Scheme of the main steps of recombinant protein production and isotopic labeling. Steps from bacteria transformation to recombinant protein production are outlined as described in paragraph 1 of the protocol. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55001/55001fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/55001/55001fig2.jpg" /> 
Figure 2: Scheme of the main steps of recombinant Tau protein purification. Steps from bacterial cells lysis to recombinant protein purification are outlined as described in paragraph 2 of the protocol. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55001/55001fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/55001/55001fig3.jpg" /> 
Figure 3: Liquid chromatography steps of protocol. (A) Cation exchange chromatography fractionation of the heated bacterial extract. The absorbance at 280 nm, 260 nm and the conductivity correspond respectively to solid and dashed black lines and dotted red line. 12% SDS-PAGE analysis of the collected fractions is shown above the chromatogram. (B) Desalting of the Tau protein into a buffer suitable for lyophilization. The amount of purified Tau protein estimated from the peak area (2,260 mAU*ml) is 16 mg of Tau. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55001/55001fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/55001/55001fig4.jpg" /> 
Figure 4: 12% SDS-PAGE analysis of Tau. Lane 1, molecular weight marker; lane 2, 10 &#181;g of Tau; lane 3, 10 &#181;g of ERK-phosphorylated Tau. Tau, as other IDPs, runs in an anomalous manner on SDS-PAGE, at an apparent molecular weight of about 60 kDa instead of the expected 46 kDa. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55001/55001fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/55001/55001fig5.jpg" /> 
Figure 5: Scheme of the main steps for NMR sample preparation, NMR spectroscopy data acquisition and data processing. Steps from NMR sample preparation to data acquisition and processing are outlined as described in paragraph 4 of the protocol. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55001/55001fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/55001/55001fig6.jpg" /> 
Figure 6: Set-up of the p1 parameter for NMR data acquisition. This parameter differs between samples and is mainly dependent on salt concentration. A standard 1H nutation curve for 80% H2O in D2O is shown. Single-scan spectra with a recycle delay of 30 sec were collected and plotted horizontally. The pulse (p1) was varied from 1 &#181;sec to 55 &#181;sec in steps of 1 &#181;sec. In theory, the signal should be maximal for a 90&#176; pulse and equal to zero for a 180&#176; pulse. However, in practice, radiation damping causes asymmetry and phase distortion problems which make it difficult to determine the 90&#176; or 180&#176; pulses directly. The second null point corresponds to a 360&#176; pulse duration. The enlarged region shows a residual signal for a 360&#176; pulse that is used to define the o1p frequency parameter. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55001/55001fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 7" src="/files/ftp_upload/55001/55001fig7.jpg" /> 
Figure 7: NMR data processing. (A) Free induction signal decay in the time domain. 1D proton spectra (B) resulting from Fourier transformation of the FID from panel A into the frequency domain but with incorrect phase (PHC0 -206&#176;). (C) phased (PHC0 -113&#176;) and referenced (TMSP signal at 0 ppm). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55001/55001fig7large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 8" src="/files/ftp_upload/55001/55001fig8.jpg" /> 
Figure 8: 2D 1H,15N HSQC spectra of recombinant 15N-Tau at 900 MHz. 3,072 and 416 acquisition data points were recorded using spectral widths of 14 and 25 ppm in the 1H (F2) and 15N (F1) dimensions, respectively. 16 scans were recorded per F1 increment, leading to a duration of the experiment of 4 hr 30 min. (A) good quality Tau sample (B) Tau sample showing degradation as revealed by the appearance of additional resonances in a particular region of the spectrum (high field 1H, low field 15N), here boxed in blue. This last sample was prepared without protease inhibitors. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55001/55001fig8large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 9" src="/files/ftp_upload/55001/55001fig9.jpg" /> 
Figure 9: 2D 1H,15N HSQC spectra of recombinant 15N-Tau. (A) unphosphorylated Tau, 600 MHz spectrum (B) phosphorylated Tau, 600 MHz spectrum and (C) phosphorylated Tau, 900 MHz spectrum. Additional resonances in a particular region of the spectrum, here boxed in red, are observed in phosphorylated Tau spectra. These resonances, which correspond to proton amide (1H-15N) correlations of pSer and pThr residues, are easily visualized in the region around 8.5 to 9.5 ppm for 1H and 117 to 125 ppm for 15N, outside the bulk of the 1H, 15N correlations of the unphosphorylated Tau spectrum. (A and B) correspond to spectra acquired at 600 MHz, 2,048 and 256 data points at spectral widths of 14 and 25 ppm were recorded in the 1H (F2) and 15N (F1) dimensions, respectively. 32 scans were used, and total duration of the acquisition was 2 hr 44 min and (C) at 900 MHz, 3,072 and 416 data points at spectral widths of 14 and 25 ppm were recorded in the 1H (F2) and 15N (F1) dimensions, respectively. 48 scans were used, and total duration of the acquisition was 6 hr 37 min. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55001/55001fig9large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 10" src="/files/ftp_upload/55001/55001fig10.jpg" /> 
Figure 10: 3D 1H,15N,13C NMR spectrum, projections and extracted 2D planes. 2,048, 72, and 256 data points were recorded in the 1H (F3), 15N (F2), and 13C (F1) dimensions, respectively. Spectral widths are 14, 25, and 61 ppm, centered on 4.7, 119, and 41 ppm in the 1H, 15N and 13C dimensions, respectively. Duration of the acquisition using 16 scans is 4 days and 6 hr. (A) Cube representation corresponding to the Fourier transformed 3D dataset of an HNCACB spectrum of ERK-phosphorylated Tau obtained at 600 MHz. The 2D 1H, 15N and the 1H, 13C planes are obtained by projection of the 3D data along the 13C and 15N dimension, respectively. Data processing and representation were done using NMR acquisition and processing software. (B) 2D 1H, 13C plane extracted from the 3D 1H,15N,13C NMR dataset at a 15N chemical shift of 121.8 ppm. A zoom (on the right) centered on the 1H chemical shift of 9.38 ppm shows the 13CA and 13CB resonances of both residue i (pThr153) and i-1 (Ala152). The 13CB resonance is aliased due to the width of the spectral window. Graphical representation and peak picking were performed using NMR analysis software. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55001/55001fig10large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins at JoVE.com

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

We describe here a method to identify multiple phosphorylations of an intrinsically disordered protein by Nuclear Magnetic Resonance Spectroscopy (NMR), using Tau protein as a case study. Recombinant Tau is isotopically enriched and modified in vitro by a kinase prior to data acquisition and analysis.

Aggregates of the neuronal Tau protein are found inside neurons of Alzheimer's disease patients. Development of the disease is accompanied by increased, ...

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18.7K Views.  Lille University. We describe here a method to identify multiple phosphorylations of an intrinsically disordered protein by Nuclear Magnetic Resonance Spectroscopy (NMR), using Tau protein as a case study. Recombinant Tau is isotopically enriched and modified in vitro by a kinase prior to data acquisition and analysis.

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

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Oligopeptide Competition Assay for Phosphorylation Site Determination

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects biological functions and characteristics of the cell. Currently, the most common method for discovering phosphorylation sites is by liquid chromatography/mass spectrometry (LC/MS) analysis, a rapid and sensitive method. However, relatively labile phosphate moieties are often released from phosphopeptides during the fragmentation step, which often yields false-negative signals. In such cases, a traditional in vitro kinase assay using site-directed mutants would be more accurate, but this method is laborious and time-consuming. Therefore, an alternative method using peptide competition may be advantageous. The consensus recognition motif of 5&#39; adenosine monophosphate-activated protein kinase (AMPK) has been established1 and was validated using a positional scanning peptide library assay2. Thus, AMPK phosphorylation sites for a novel substrate could be predicted and confirmed by the peptide competition assays. In this report, we describe the detailed steps and procedures for the in vitro oligopeptide-competing kinase assay by illustrating AMPK-mediated nuclear factor erythroid 2-related factor 2 (Nrf2) phosphorylation. To authenticate the phosphorylation site, we carried out a sequential in vitro kinase assay using a site-specific mutant. Overall, the peptide competition assay provides a method to screen multiple potential phosphorylation sites and to identify sites for validation by the phosphorylation site mutants.

Peptide competition assays are widely used in a variety of molecular and immunological experiments. This paper describes a detailed method for an in vitro oligopeptide-competing kinase assay and the associated validation procedures, which may be useful to find specific phosphorylation sites.

Protein phosphorylation at specific sites determines its conformation and interaction with other molecules. Thus, protein phosphorylation affects ...

Atomic Scale Structural Studies of Macromolecular Assemblies by Solid-state Nuclear Magnetic Resonance Spectroscopy

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the propagation of neurodegenerative disorders. Such assemblies consist in multiple protein subunits organized in a non-covalent way to form large macromolecular objects that can execute a variety of cellular functions or cause detrimental consequences. Atomic insights into the assembly mechanisms and the functioning of those macromolecular assemblies remain often scarce since their inherent insolubility and non-crystallinity often drastically reduces the quality of the data obtained from most techniques used in structural biology, such as X-ray crystallography and solution Nuclear Magnetic Resonance (NMR). We here present magic-angle spinning solid-state NMR spectroscopy (SSNMR) as a powerful method to investigate structures of macromolecular assemblies at atomic resolution. SSNMR can reveal atomic details on the assembled complex without size and solubility limitations. The protocol presented here describes the essential steps from the production of 13C/15N isotope-labeled macromolecular protein assemblies to the acquisition of standard SSNMR spectra and their analysis and interpretation. As an example, we show the pipeline of a SSNMR structural analysis of a filamentous protein assembly.

Structures of supramolecular protein assemblies at atomic resolution are of high relevance because of their crucial roles in a variety of biological phenomena. Herein, we present a protocol to perform high-resolution structural studies on insoluble and non-crystalline macromolecular protein assemblies by magic-angle spinning solid-state nuclear magnetic resonance spectroscopy (MAS SSNMR).

Supramolecular protein assemblies play fundamental roles in biological processes ranging from host-pathogen interaction, viral infection to the ...

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; however, the number of identified targets for Cdk1, particularly in human cells is still low. The identification of Cdk1-specific phosphorylation sites is important, as they provide mechanistic insights into how Cdk1 controls the cell cycle. Cell cycle regulation is critical for faithful chromosome segregation, and defects in this complicated process lead to chromosomal aberrations and cancer.
Here, we describe an in vitro kinase assay that is used to identify Cdk1-specific phosphorylation sites. In this assay, a purified protein is phosphorylated in vitro by commercially available human Cdk1/cyclin B. Successful phosphorylation is confirmed by SDS-PAGE, and phosphorylation sites are subsequently identified by mass spectrometry. We also describe purification protocols that yield highly pure and homogeneous protein preparations suitable for the kinase assay, and a binding assay for the functional verification of the identified phosphorylation sites, which probes the interaction between a classical nuclear localization signal (cNLS) and its nuclear transport receptor karyopherin &#945;. To aid with experimental design, we review approaches for the prediction of Cdk1-specific phosphorylation sites from protein sequences. Together these protocols present a very powerful approach that yields Cdk1-specific phosphorylation sites and enables mechanistic studies into how Cdk1 controls the cell cycle. Since this method relies on purified proteins, it can be applied to any model organism and yields reliable results, especially when combined with cell functional studies.

Identification of Cyclin-dependent Kinase 1 Specific Phosphorylation Sites by an In Vitro Kinase Assay

Cyclin-dependent kinase 1 (Cdk1) is activated in the G2 phase of the cell cycle and regulates many cellular pathways. Here, we present a protocol for an in vitro kinase assay with Cdk1, which allows the identification of Cdk1-specific phosphorylation sites for establishing cellular targets of this important kinase.

Cyclin-dependent kinase 1 (Cdk1) is a master controller for the cell cycle in all eukaryotes and phosphorylates an estimated 8 - 13% of the proteome; ...

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Biology

Paramagnetic Relaxation Enhancement for Detecting and Characterizing Self-Associations of Intrinsically Disordered Proteins

Intrinsically disordered proteins and intrinsically disordered regions within proteins make up a large and functionally significant part of the human proteome. The highly flexible nature of these sequences allows them to form weak, long-range, and transient interactions with diverse biomolecular partners. Specific yet low-affinity interactions promote promiscuous binding and enable a single intrinsically disordered segment to interact with a multitude of target sites. Because of the transient nature of these interactions, they can be difficult to characterize by structural biology methods that rely on proteins to form a single, predominant conformation. Paramagnetic relaxation enhancement NMR is a useful tool for identifying and defining the structural underpinning of weak and transient interactions. A detailed protocol for using paramagnetic relaxation enhancement to characterize the lowly-populated encounter complexes that form between intrinsically disordered proteins and their protein, nucleic acid, or other biomolecular partners is described.

A protocol for the application of paramagnetic relaxation enhancement NMR spectroscopy to detect weak and transient inter- and intra-molecular interactions in intrinsically disordered proteins is presented.

Intrinsically disordered proteins and intrinsically disordered regions within proteins make up a large and functionally significant part of the human ...

An Optimized Single-Molecule Pull-Down Assay for Quantification of Protein Phosphorylation

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to measure protein phosphorylation cannot preserve the heterogeneity in phosphorylation across individual proteins. The single-molecule pull-down (SiMPull) assay was developed to investigate the composition of macromolecular complexes via immunoprecipitation of proteins on a glass coverslip followed by single-molecule imaging. The current technique is an adaptation of SiMPull that provides robust quantification of the phosphorylation state of full-length membrane receptors at the single-molecule level. Imaging thousands of individual receptors in this way allows for quantifying protein phosphorylation patterns. The present protocol details the optimized SiMPull procedure, from sample preparation to imaging. Optimization of glass preparation and antibody fixation protocols further enhances data quality. The current protocol provides code for the single-molecule data analysis that calculates the fraction of receptors phosphorylated within a sample. While this work focuses on phosphorylation of the epidermal growth factor receptor (EGFR), the protocol can be generalized to other membrane receptors and cytosolic signaling molecules.

The present protocol describes sample preparation and data analysis to quantify protein phosphorylation using an improved single-molecule pull-down (SiMPull) assay.

Phosphorylation is a necessary posttranslational modification that regulates protein function and directs cell signaling outcomes. Current methods to ...

Protocol for NMR 15N Relaxation and hetNOE Experiments in Protein Dynamics Study

NMR 15N Relaxation Experiments for the Investigation of Picosecond to Nanoseconds Structural Dynamics of Proteins

Nuclear magnetic resonance (NMR) spectroscopy allows studying proteins in solution and under physiological temperatures. Frequently, either the amide groups of the protein backbone or the methyl groups in side chains are used as reporters of structural dynamics in proteins. A structural dynamics study of the protein backbone of globular proteins on 15N labeled and fully protonated samples usually works well for proteins with a molecular weight of up to 50 kDa. When side chain deuteration in combination with transverse relaxation optimized spectroscopy (TROSY) is applied, this limit can be extended up to 200 kDa for globular proteins and up to 1 MDa when the focus is on the side chains. When intrinsically disordered proteins (IDPs) or proteins with intrinsically disordered regions (IDRs) are investigated, these weight limitations do not apply but can go well beyond. The reason is that IDPs or IDRs, characterized by high internal flexibility, are frequently dynamically decoupled. Various NMR methods offer atomic-resolution insights into structural protein dynamics across a wide range of time scales, from picoseconds up to hours. Standard 15N relaxation measurements overview a protein&#39;s internal flexibility and characterize the protein backbone dynamics experienced on the fast pico- to nanosecond timescale. This article presents a hands-on protocol for setting up and recording NMR 15N R1, R2, and heteronuclear Overhauser effect (hetNOE) experiments. We show exemplary data and explain how to interpret them simply qualitatively before any more sophisticated analysis.

Author Spotlight: Exploring Intrinsically Disordered Protein Dynamics Through NMR Relaxation Experiments

Nuclear magnetic resonance (NMR) spectroscopy can characterize structural protein dynamics in a residue-specific manner. We provide a hands-on protocol for recording NMR 15N R1 and R2 relaxation and {1H}-15N heteronuclear Overhauser effect (hetNOE) experiments, sensitive to the picoseconds to nanoseconds timescale.

Nuclear magnetic resonance (NMR) spectroscopy allows studying proteins in solution and under physiological temperatures. Frequently, either the amide ...

Identification of Post-translational Modifications of Plant Protein Complexes

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to infection via the recruitment and activation of immune complexes. Activation of immune complexes is associated with post-translational modifications (PTMs) of proteins, such as phosphorylation, glycosylation, or ubiquitination. Understanding how these PTMs are choreographed will lead to a better understanding of how resistance is achieved.
Here we describe a protein purification method for nucleotide-binding leucine-rich repeat (NB-LRR)-interacting proteins and the subsequent identification of their post-translational modifications (PTMs). With small modifications, the protocol can be applied for the purification of other plant protein complexes. The method is based on the expression of an epitope-tagged version of the protein of interest, which is subsequently partially purified by immunoprecipitation and subjected to mass spectrometry for identification of interacting proteins and PTMs.
This protocol demonstrates that: i). Dynamic changes in PTMs such as phosphorylation can be detected by mass spectrometry; ii). It is important to have sufficient quantities of the protein of interest, and this can compensate for the lack of purity of the immunoprecipitate; iii). In order to detect PTMs of a protein of interest, this protein has to be immunoprecipitated to get a sufficient quantity of protein.

We describe here a protocol for the purification and characterization of plant protein complexes. We demonstrate that by immunoprecipitating a single protein within a complex, so we can identify its post-translational modifications and its interacting partners.

Plants adapt quickly to changing environments due to elaborate perception and signaling systems. During pathogen attack, plants rapidly respond to ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...

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

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