title: Nuclear Magnetic Resonance Spectroscopy for the Identification of Multiple Phosphorylations of Intrinsically Disordered Proteins.
abstract: 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.
authors.affiliation: UMR8576, CNRS, Lille University; UMR-S1172, INSERM CNRS, Lille University.
authors.forename: Clément
authors.initials: C
authors.lastname: Danis
authors.affiliation: UMR8576, CNRS, Lille University.
authors.forename: Clément
authors.initials: C
authors.lastname: Despres
authors.affiliation: UMR8576, CNRS, Lille University.
authors.forename: Luiza M
authors.initials: LM
authors.lastname: Bessa
authors.affiliation: UMR8576, CNRS, Lille University.
authors.forename: Idir
authors.initials: I
authors.lastname: Malki
authors.affiliation: UMR8576, CNRS, Lille University.
authors.forename: Hamida
authors.initials: H
authors.lastname: Merzougui
authors.affiliation: UMR8576, CNRS, Lille University.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Huvent
authors.affiliation: UMR8576, CNRS, Lille University.
authors.forename: Haoling
authors.initials: H
authors.lastname: Qi
authors.affiliation: UMR8576, CNRS, Lille University.
authors.forename: Guy
authors.initials: G
authors.lastname: Lippens
authors.affiliation: UMR8576, CNRS, Lille University.
authors.forename: François-Xavier
authors.initials: FX
authors.lastname: Cantrelle
authors.affiliation: UMR8576, CNRS, Lille University.
authors.forename: Robert
authors.initials: R
authors.lastname: Schneider
authors.affiliation: UMR8576, CNRS, Lille University.
authors.forename: Xavier
authors.initials: X
authors.lastname: Hanoulle
authors.affiliation: UMR8576, CNRS, Lille University.
authors.forename: Caroline
authors.initials: C
authors.lastname: Smet-Nocca
authors.affiliation: UMR8576, CNRS, Lille University; isabelle.landrieu@univ-lille1.fr.
authors.forename: Isabelle
authors.initials: I
authors.lastname: Landrieu
mesh_terms: D000544:Alzheimer Disease; D006801:Humans; D064267:Intrinsically Disordered Proteins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D010766:Phosphorylation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D016875:tau Proteins / Q000737:chemistry*
chemical_list: D064267:Intrinsically Disordered Proteins; D011994:Recombinant Proteins; D016875:tau Proteins