Probing exchange kinetics and atomic resolution dynamics in high-molecular-weight complexes using dark-state exchange saturation transfer NMR spectroscopy.
Probing exchange kinetics and atomic resolution dynamics in high-molecular-weight complexes using dark-state exchange saturation transfer NMR spectroscopy.
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DOI：
10.1038/nprot.2012.077
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发表时间：
2012-07-19
期刊：
影响因子：
14.8
通讯作者：
Clore, G. Marius
中科院分区：
文献类型：
--
作者：
Fawzi, Nicolas L.;Ying, Jinfa;Torchia, Dennis A.;Clore, G. Marius
我们提出了暗态交换饱和转移（DEST）的测量和分析的协议，一种新的溶液NMR方法，用于表征，在原子分辨率，NMR-“可见”的自由物种和NMR-“不可见”的物种之间的相互作用瞬时结合到一个非常高的分子量（>1 MDa）的大分子实体。在束缚态的重定向运动，排除了传统的NMR方法的表征率降低允许观察的DEST。在包含与NMR可见物质交换的暗态的样品上测量15 N-DEST曲线;此外，还获得该样品与仅包含NMR可见物质的对照样品之间的15 N横向弛豫速率的差异（ΔR2）。然后将所有残基的15 N-DEST和ΔR2数据同时拟合到各种交换模型的McConnell方程，这些模型描述了结合态下的残基特异性动力学和相互转化速率常数。尽管实验的长度在很大程度上取决于样品条件，但大约1周的NMR光谱仪时间足以对浓度约为100 μM的β淀粉样蛋白（Aβ）样品进行全面表征。
We present the protocol for the measurement and analysis of dark-state exchange saturation transfer (DEST), a novel solution NMR method for characterizing, at atomic resolution, the interaction between an NMR-‘visible’ free species and an NMR-‘invisible’ species transiently bound to a very high-molecular-weight (>1 MDa) macromolecular entity. The reduced rate of reorientational motion in the bound state that precludes characterization by traditional NMR methods permits the observation of DEST. 15N-DEST profiles are measured on a sample comprising the dark state in exchange with an NMR-visible species; in addition, the difference (ΔR2) in 15N transverse relaxation rates between this sample and a control sample comprising only the NMR-visible species is also obtained. The 15N-DEST and ΔR2 data for all residues are then fitted simultaneously to the McConnell equations for various exchange models describing the residue-specific dynamics in the bound state(s) and the interconversion rate constants. Although the length of the experiments depends strongly on sample conditions, approximately 1 week of NMR spectrometer time was sufficient for full characterization of samples of amyloid-β (Aβ) at concentrations of ~100 μM.
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影响因子：
64.8
作者：
RODER, H;ELOVE, GA;ENGLANDER, SW
通讯作者：
ENGLANDER, SW
影响因子：
15
作者：
Fawzi, Nicolas L.;Ying, Jinfa;Torchia, Dennis A.;Clore, G. Marius
通讯作者：
Clore, G. Marius
影响因子：
62.1
作者：
Clore, G. Marius;Iwahara, Junji
通讯作者：
Iwahara, Junji
影响因子：
5.6
作者：
Bodner, Christina R.;Dobson, Christopher M.;Bax, Ad
通讯作者：
Bax, Ad
影响因子：
2.2
作者：
CLORE, GM;GRONENBORN, AM
通讯作者：
GRONENBORN, AM