利用溶解 DNP NMR 光谱实时分析无规卷曲蛋白结合时的折叠情况。
Real-Time Analysis of Folding upon Binding of a Disordered Protein by Using Dissolution DNP NMR Spectroscopy.
机构信息
Department of Biochemistry & Biophysics, Texas A&M University, College Station, TX, 77843, USA.
Department of Biochemistry and Molecular Biology, University of Florida College of Medicine, Gainesville, FL, 32611, USA.
出版信息
Angew Chem Int Ed Engl. 2017 Jun 12;56(25):7070-7073. doi: 10.1002/anie.201700464. Epub 2017 May 16.
Abstract
The kinase inhibitory domain of the cell cycle regulatory protein p27 (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 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.
摘要
细胞周期调控蛋白 p27 的激酶抑制结构域（p27）使用溶解动态核极化（D-DNP）实现核自旋超极化。p27 在与细胞周期蛋白依赖性激酶 2（Cdk2）/细胞周期蛋白 A 结合时，虽然在单独存在时是无规则卷曲的，但会采用二级结构模体，包括α-螺旋结构。通过极化获得的灵敏度提高使得能够在 p27 与 Cdk2/细胞周期蛋白 A 结合时，在几秒钟的时间尺度内实时观察到 C-NMR 信号的折叠。时间依赖性强度变化取决于折叠和结合的程度，表现在不同的自旋弛豫上。信号衰减速率的分析表明，在 D-DNP NMR 实验的时间尺度内存在部分折叠的 p27 中间产物。
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