Measuring the Signs of 1Hα Chemical Shift Differences Between Ground and Excited Protein States by Off-Resonance Spin-Lock R1ρ NMR Spectroscopy
Measuring the Signs of 1Hα Chemical Shift Differences Between Ground and Excited Protein States by Off-Resonance Spin-Lock R1ρ NMR Spectroscopy
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DOI：
10.1021/ja904315m
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发表时间：
2009-08-12
影响因子：
15
通讯作者：
Kay, Lewis E.
中科院分区：
文献类型：
--
作者：
Auer, Renate;Neudecker, Philipp;Kay, Lewis E.
Carr-Purcell-Meiboom-Gill（CPMG）弛豫色散NMR曲线的分析提供了毫秒时间尺度交换过程的动力学和热力学，涉及填充基态和不可见激发态的相互转换。此外，还提取了交换状态下NMR探针之间的化学位移差的绝对值，垂直棒Delta（pi）垂直棒。在这里，我们提出了一个简单的实验，通过测量非共振H-1（α）衰变率，R-1 ρ，使用弱质子自旋锁定场获得的H-1（α）δ（π）值的符号。一对R-1 ρ值用施加自旋锁定场的垂直条Δ ω垂直条测量主状态峰的低场和高场。在许多情况下，这两个弛豫速率有很大的不同，较大的弛豫速率对应于自旋锁场与处于次要状态的探针的共振频率一致的情况。该方法的实用性首先在涉及蛋白质配体交换的系统上得到证明，随后在折叠状态和其通路折叠中间体之间的SH 3结构域交换上得到证明。通过这个实验，可以确定看不见的激发态的H-1（α）化学位移，这可以用作定义这些难以捉摸的构象的结构特性的强大约束。
Analysis of Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion NMR profiles provides the kinetics and thermodynamics of millisecond-time-scale exchange processes involving the interconversion of populated ground and invisible excited states. In addition, the absolute values of chemical, shift differences between NMR probes in the exchanging states, vertical bar Delta(pi)vertical bar, are also extracted. Herein, we present a simple experiment for obtaining the sign of H-1(alpha) Delta(pi) values by measuring off-resonance H-1(alpha) decay rates, R-1 rho, using weak proton spin-lock fields. A pair of R-1 rho values is measured with a spin-lock field applied vertical bar Delta omega vertical bar downfield and upfield of the major-state peak. In many cases, these two relaxation rates differ substantially, with the larger one corresponding to the case where the spin-lock field coincides with the resonance frequency of the probe in the minor state. The utility of the methodology is demonstrated first on a system involving protein ligand exchange and subsequently on an SH3 domain exchanging between a folded state and its on-pathway folding intermediate. With this experiment, it thus becomes possible to determine H-1(alpha) chemical shifts of the invisible excited state, which can be used as powerful restraints in defining the structural properties of these elusive conformers.