3 years ago

Real-Time Analysis of Folding upon Binding of a Disordered Protein by using Dissolution DNP NMR Spectroscopy

Real-Time Analysis of Folding upon Binding of a Disordered Protein by using Dissolution DNP NMR Spectroscopy
Mukundan Ragavan, Luigi I. Iconaru, Cheon-Gil Park, Richard W. Kriwacki, Christian Hilty
The kinase inhibitory domain of the cell cycle regulatory protein p27Kip1 (p27) was nuclear spin hyperpolarized using dissolution dynamic nuclear polarization (D-DNP). While intrinsically disordered in isolation, p27 adopts secondary structural motifs, including an α-helical structure, upon binding to cyclin-dependent kinase 2 (Cdk2)/cyclin A. The sensitivity gains obtained with hyperpolarization enable the real-time observation of 13C NMR signals during p27 folding upon binding to Cdk2/cyclin A on a time scale of several seconds. Time-dependent intensity changes are dependent on the extent of folding and binding, as manifested in differential spin relaxation. The analysis of signal decay rates suggests the existence of a partially folded p27 intermediate during the timescale of the D-DNP NMR experiment. Protein–protein interactions on the fly: Intrinsically disordered protein p27Kip1 folds upon binding to Cdk2/cyclin A. Real-time 13C NMR spectroscopy is enabled on the folding time scale using hyperpolarization through dissolution dynamic nuclear polarization. The time evolution of the signals suggests that a partially folded intermediate can be observed.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/anie.201700464

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