Measurement of carbonyl chemical shifts of excited protein states by relaxation dispersion NMR spectroscopy:: comparison between uniformly and selectively 13C labeled samples
Measurement of carbonyl chemical shifts of excited protein states by relaxation dispersion NMR spectroscopy:: comparison between uniformly and selectively 13C labeled samples
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DOI：
10.1007/s10858-008-9260-4
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发表时间：
2008-09-01
影响因子：
2.7
通讯作者：
Kay, Lewis E.
中科院分区：
文献类型：
--
作者：
Lundstroem, Patrik;Hansen, D. Flemming;Kay, Lewis E.
Carr-Purcell-Meiboom-Gill (CPMG) 弛豫色散核磁共振 (NMR) 光谱已成为量化激发蛋白质态化学位移的强大方法。对于涉及测量碳磁化弛豫率的该技术的许多应用，有必要制备具有孤立的 C-13 自旋的样品，以便实验不会遭受耦合碳自旋之间的磁化转移，否则在 CPMG 脉冲序列期间会发生这种情况。然而，在 (CO)-C-13 实验中，(CO)-C-13 和 C-13(α) 化学位移之间的巨大分离提供了希望，可以在均匀的 C-13 标记样品上记录稳健的 (CO)-C-13 分散曲线，从而提取不可见激发态的准确 (CO)-C-13 化学位移。在这里，我们比较了使用 [1-C-13]-丙酮酸和 (NaHCO3,)-C-13 制备的选择性标记的样品或由 C-13-葡萄糖生成的统一标记的样品上记录的化学位移。通过对两个样品上记录的 CPMG 实验的分析获得了非常相似的 (CO)-C-13 化学位移，并且与使用第二种方法测量的化学位移进行比较表明，根据弛豫色散测量的位移非常准确。
Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion nuclear magnetic resonance (NMR) spectroscopy has emerged as a powerful method for quantifying chemical shifts of excited protein states. For many applications of the technique that involve the measurement of relaxation rates of carbon magnetization it is necessary to prepare samples with isolated C-13 spins so that experiments do not suffer from magnetization transfer between coupled carbon spins that would otherwise occur during the CPMG pulse train. In the case of (CO)-C-13 experiments however the large separation between (CO)-C-13 and C-13(alpha) chemical shifts offers hope that robust (CO)-C-13 dispersion profiles can be recorded on uniformly C-13 labeled samples, leading to the extraction of accurate (CO)-C-13 chemical shifts of the invisible, excited state. Here we compare such chemical shifts recorded on samples that are selectively labeled, prepared using [1-C-13]-pyruvate and (NaHCO3,)-C-13 or uniformly labeled, generated from C-13-glucose. Very similar (CO)-C-13 chemical shifts are obtained from analysis of CPMG experiments recorded on both samples, and comparison with chemical shifts measured using a second approach establishes that the shifts measured from relaxation dispersion are very accurate.