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 13C and 15N stable isotopes to enable the direct recording of 13C-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.
In-cell 13C NMR spectroscopy for the study of intrinsically disordered proteins / Isabella C Felli;Leonardo Gonnelli;Roberta Pierattelli. - In: NATURE PROTOCOLS. - ISSN 1754-2189. - ELETTRONICO. - 9:(2014), pp. 2005-2016. [10.1038/nprot.2014.124]
In-cell 13C NMR spectroscopy for the study of intrinsically disordered proteins
FELLI, ISABELLA CATERINA;GONNELLI, LEONARDO;PIERATTELLI, ROBERTA
2014
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 13C and 15N stable isotopes to enable the direct recording of 13C-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.I documenti in FLORE sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.