title: In-cell ¹³C NMR spectroscopy for the study of intrinsically disordered proteins.
abstract: A large number of proteins carry out their function in highly flexible and disordered states, lacking a well-defined 3D structure. These proteins, referred to as intrinsically disordered proteins (IDPs), are now in the spotlight of modern structural biology. Nuclear magnetic resonance (NMR) spectroscopy represents a unique tool for accessing atomic resolution information on IDPs in complex environments as whole cells, provided that the methods are optimized to their peculiar properties and to the characteristics of in-cell experiments. We describe procedures for the preparation of in-cell NMR samples, as well as for the setup of NMR experiments and their application to in-cell studies, using human α-synuclein overexpressed in Escherichia coli as an example. The expressed protein is labeled with (13)C and (15)N stable isotopes to enable the direct recording of (13)C-detected NMR experiments optimized for the properties of IDPs. The entire procedure covers 24 h, including cell transformation, cell growth overnight, setup of the spectrometer and NMR experiment recording.
authors: Magnetic Resonance Center (CERM), Department of Chemistry 'Ugo Schiff', University of Florence, Sesto Fiorentino, Italy.
authors: Isabella C
authors: IC
authors: Felli
authors: Magnetic Resonance Center (CERM), Department of Chemistry 'Ugo Schiff', University of Florence, Sesto Fiorentino, Italy.
authors: Leonardo
authors: L
authors: Gonnelli
authors: Magnetic Resonance Center (CERM), Department of Chemistry 'Ugo Schiff', University of Florence, Sesto Fiorentino, Italy.
authors: Roberta
authors: 0000000177550885
authors: R
authors: Pierattelli
mesh_terms: D002247:Carbon Isotopes; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D004926:Escherichia coli; D006801:Humans; D064267:Intrinsically Disordered Proteins / Q000032:analysis*; D015394:Molecular Structure; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D012680:Sensitivity and Specificity; D051844:alpha-Synuclein / Q000032:analysis
chemical_list: D002247:Carbon Isotopes; D064267:Intrinsically Disordered Proteins; D009587:Nitrogen Isotopes; D051844:alpha-Synuclein