Name: nuclear magnetic resonance spectroscopy for the identification of multiple phosphorylations of intrinsically disordered proteins
Uploaded: 2016-12-27T14:00:00
Description: Watch this Scientific Journal Video about Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins at JoVE.com

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 ...

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 th...

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.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">pET15B recombinant T7 expression plasmid</td>
			<td style="width:177px;">Novagen</td>
			<td style="width:119px;">69257</td>
			<td style="width:196px;">Keep at -20&#176;C</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:328px;">BL21(DE3) transformation competent E.coli bacteria</td>
			<td style="width:177px;">New England Biolabs</td>
			<td style="width:119px;">C2527I</td>
			<td style="width:196px;">Keep at -80&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Autoclaved LB Broth, Lennox&#160;</td>
			<td style="width:177px;">DIFCO</td>
			<td style="width:119px;">240210</td>
			<td style="width:196px;">Bacterial Growth Medium</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:328px;">MEM vitamin complements 100X</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:119px;">58970C</td>
			<td style="width:196px;">Bacterial Growth Medium Supplement</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:328px;">15N, 13C-ISOGRO complete medium powder</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:119px;">608297</td>
			<td style="width:196px;">Bacterial Growth Medium Supplement</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:328px;">15NH4Cl</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:119px;">299251</td>
			<td style="width:196px;">Isotope</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:328px;">13C6-Glucose</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:119px;">389374</td>
			<td style="width:196px;">Isotope</td>
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		<tr height="20">
			<td height="61" rowspan="2" style="height:61px;width:328px;">Protease inhibitor tablets&#160;</td>
			<td rowspan="2" style="width:177px;">Roche</td>
			<td rowspan="2" style="width:119px;">5056489001</td>
			<td style="width:196px;">Keep at 4&#176;C</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:196px;">1 tablet in 1ml is 40X solution that can be kept at -20&#176;C</td>
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		<tr height="21">
			<td height="21" style="height:21px;width:328px;">DNaseI</td>
			<td style="width:177px;">EUROMEDEX</td>
			<td style="width:119px;">1307</td>
			<td style="width:196px;">Keep at -20&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Homogenizer (EmulsiFlex-C3)</td>
			<td style="width:177px;">AVESTIN</td>
			<td style="width:119px;"></td>
			<td style="width:196px;">Lysis is realized at 4&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Pierce&#8482; Unstained Protein MW Marker</td>
			<td style="width:177px;">Pierce</td>
			<td style="width:119px;">266109</td>
			<td style="width:196px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Active human MEK1 kinase, GST Tagged</td>
			<td style="width:177px;">Sigma</td>
			<td style="width:119px;">M8822</td>
			<td style="width:196px;">Keep at -80&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">AKT&#196; Pure chromatography system</td>
			<td style="width:177px;">GE Healthcare</td>
			<td style="width:119px;"></td>
			<td style="width:196px;">FPLC</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:328px;">HiTrap SP Sepharose FF (5 mL column)</td>
			<td style="width:177px;">GE Healthcare</td>
			<td style="width:119px;">17-5156-01</td>
			<td style="width:196px;">Cation exchange chromatography columns</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:328px;">HiPrep 26/10 Desalting</td>
			<td style="width:177px;">GE Healthcare</td>
			<td style="width:119px;">17-5087-01</td>
			<td>Protein Desalting column</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:328px;">PD MidiTrap G-25</td>
			<td style="width:177px;">GE Healthcare</td>
			<td style="width:119px;">28-9180-08</td>
			<td style="width:196px;">Protein Desalting column</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">Tris D11, 97% D</td>
			<td style="width:177px;">Cortecnet</td>
			<td>CD4035P5</td>
			<td style="width:196px;">Deuterated NMR buffer</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:328px;">5 mm Symmetrical Microtube SHIGEMI D2O ( set of 5 inner &#38; outerpipe)&#160;</td>
			<td style="width:177px;">Euriso-top</td>
			<td style="width:119px;">BMS-005B</td>
			<td style="width:196px;">NMR Shigemi Tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:328px;">eVol kit-electronic syringe starter kit</td>
			<td style="width:177px;">Cortecnet</td>
			<td style="width:119px;">2910000</td>
			<td style="width:196px;">Pipetting</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:328px;">Bruker 900MHz AvanceIII with a triple resonance cryogenic probehead</td>
			<td style="width:177px;">Bruker</td>
			<td style="width:119px;"></td>
			<td style="width:196px;">NMR spectrometer for data acquisition</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:328px;">Bruker 600MHz DMX600 with a triple resonance cryogenic probehead</td>
			<td style="width:177px;">Bruker</td>
			<td style="width:119px;"></td>
			<td style="width:196px;">NMR spectrometer for data acquisition</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:328px;">TopSpin 3.1</td>
			<td style="width:177px;">Bruker</td>
			<td style="width:119px;"></td>
			<td style="width:196px;">Acquisition and Processing software for NMR experiments</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:328px;">Sparky 3.114</td>
			<td style="width:177px;">UCSF (T. D. Goddard and D. G. Kneller)</td>
			<td style="width:119px;"></td>
			<td style="width:196px;">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</...

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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