Off-resonance TROSY-selected R1p experiment with improved sensitivity for medium- and high-molecular-weight proteins
Off-resonance TROSY-selected R1p experiment with improved sensitivity for medium- and high-molecular-weight proteins
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DOI：
10.1021/ja061692f
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发表时间：
2006-06-28
影响因子：
15
通讯作者：
Palmer, Arthur G., III
中科院分区：
文献类型：
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作者：
Igumenova, Tatyana I.;Palmer, Arthur G., III
利用弛豫干涉现象（TROSY）的NMR自旋弛豫技术能够在高分子量蛋白质中表征化学交换过程。TROSY选择（TS）方法用于测量自旋锁定旋转参考系中的非共振R1 ρ弛豫，该方法使用三个原理：（i）不可交换的1H位点的氘化，以使远程偶极-偶极相互作用最小化，（ii）缓慢弛豫的15 N双重态组分的选择性激发，以获得最佳初始条件，以及（iii）选择性反转15 N二重态分量之一以抑制自旋锁定期间的交叉弛豫。该方法在280 K下使用[90%-15 N，70%-2 H]泛素进行验证。TROSY-selectedR 1 ρ实验能够在微秒时间尺度上表征大型蛋白质的骨架动力学。
NMR spin relaxation techniques that utilize relaxation interference phenomena (TROSY) enable chemical exchange processes to be characterized in high-molecular-weight proteins. A TROSY-selected (TS) approach for measuring off-resonanceR1ρrelaxation in the spin-locked rotating reference frame is developed using three principles: (i) deuteration of nonexchangeable1H sites to minimize remote dipole−dipole interactions, (ii) selective excitation of the slowly relaxing15N doublet component to obtain optimal initial conditions, and (iii) selective inversion of one of the15N doublet components to suppress cross-relaxation during the spin-lock period. The method is validated using [90%-15N, 70%-2H] ubiquitin at 280 K. The TROSY-selectedR1ρexperiment enables characterization of backbone dynamics on the microsecond time scale in large proteins.