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Angew Chem Int Ed Engl. 2017 Jun 12;56(25):7070-7073. doi: 10.1002/anie.201700464. Epub 2017 May 16.

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

Author information

1
Department of Biochemistry & Biophysics, Texas A&M University, College Station, TX, 77843, USA.
2
Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, FL, 32611, USA.
3
Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN, 38105, USA.
4
Department of Microbiology, Immunology and Biochemistry, The University of Tennessee Health Science Center, Memphis, TN, 38163, USA.
5
Department of Chemistry, Texas A&M University, College Station, TX, 77843, USA.

Abstract

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 13 C 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.

KEYWORDS:

NMR spectroscopy; hyperpolarization; intrinsically disordered proteins; protein folding; protein-protein interactions

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