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, 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.
One of the main challenges of healthcare in the 21st century are neurodegenerative diseases such as Alzheimer´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'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 3β (Gsk3β), 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.
1. Production of 15N, 13C-Tau (Figure 1)
2. Purification of 15 N, 13 C-Tau (Figure 2)
3. In Vitro Phosphorylation of 15N-Tau
4. Acquisition of NMR Spectra (Figure 5)
5. Identification of Phosphorylation Sites
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 µsec). To setup the length of the pulse that will rotate 1H spin magnetization by 90°, a pulse corresponding to a 360° 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° pulse length is adequately defined. The value corresponding to a 360° rotation is then divided by 4. p1 in this experiment is 10.5 µsec. 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). 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.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
Figure 4: 12% SDS-PAGE analysis of Tau. Lane 1, molecular weight marker; lane 2, 10 µg of Tau; lane 3, 10 µg 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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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 µsec to 55 µsec in steps of 1 µsec. In theory, the signal should be maximal for a 90° pulse and equal to zero for a 180° pulse. However, in practice, radiation damping causes asymmetry and phase distortion problems which make it difficult to determine the 90° or 180° pulses directly. The second null point corresponds to a 360° pulse duration. The enlarged region shows a residual signal for a 360° pulse that is used to define the o1p frequency parameter. Please click here to view a larger version of this figure.
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°). (C) phased (PHC0 -113°) and referenced (TMSP signal at 0 ppm). Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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. Please click here to view a larger version of this figure.
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 µl (i.e. 100 µM) Tau protein. For a 3D spectrum, at least 200 µM in 300 µl 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 °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.
The authors have nothing to disclose.
The NMR facilities were funded by the Région 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.
pET15B recombinant T7 expression plasmid | Novagen | 69257 | Keep at -20°C |
BL21(DE3) transformation competent E.coli bacteria | New England Biolabs | C2527I | Keep at -80°C |
Autoclaved LB Broth, Lennox | DIFCO | 240210 | Bacterial Growth Medium |
MEM vitamin complements 100X | Sigma | 58970C | Bacterial Growth Medium Supplement |
15N, 13C-ISOGRO complete medium powder | Sigma | 608297 | Bacterial Growth Medium Supplement |
15NH4Cl | Sigma | 299251 | Isotope |
13C6-Glucose | Sigma | 389374 | Isotope |
Protease inhibitor tablets | Roche | 5056489001 | Keep at 4°C |
1 tablet in 1ml is 40X solution that can be kept at -20°C | |||
DNaseI | EUROMEDEX | 1307 | Keep at -20°C |
Homogenizer (EmulsiFlex-C3) | AVESTIN | Lysis is realized at 4°C | |
Pierce™ Unstained Protein MW Marker | Pierce | 266109 | |
Active human MEK1 kinase, GST Tagged | Sigma | M8822 | Keep at -80°C |
AKTÄ Pure chromatography system | GE Healthcare | FPLC | |
HiTrap SP Sepharose FF (5 mL column) | GE Healthcare | 17-5156-01 | Cation exchange chromatography columns |
HiPrep 26/10 Desalting | GE Healthcare | 17-5087-01 | Protein Desalting column |
PD MidiTrap G-25 | GE Healthcare | 28-9180-08 | Protein Desalting column |
Tris D11, 97% D | Cortecnet | CD4035P5 | Deuterated NMR buffer |
5 mm Symmetrical Microtube SHIGEMI D2O ( set of 5 inner & outerpipe) | Euriso-top | BMS-005B | NMR Shigemi Tubes |
eVol kit-electronic syringe starter kit | Cortecnet | 2910000 | Pipetting |
Bruker 900MHz AvanceIII with a triple resonance cryogenic probehead | Bruker | NMR spectrometer for data acquisition | |
Bruker 600MHz DMX600 with a triple resonance cryogenic probehead | Bruker | NMR spectrometer for data acquisition | |
TopSpin 3.1 | Bruker | Acquisition and Processing software for NMR experiments | |
Sparky 3.114 | UCSF (T. D. Goddard and D. G. Kneller) | NMR data Analysis software |