Direct measurements of protein backbone 15N spin relaxation rates from peak line-width using a fully-relaxed Accordion 3D HNCO experiment.
Direct measurements of protein backbone 15N spin relaxation rates from peak line-width using a fully-relaxed Accordion 3D HNCO experiment.
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DOI：
10.1016/j.jmr.2008.12.001
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发表时间：
2009-03
期刊：
影响因子：
--
通讯作者：
Tjandra N
中科院分区：
文献类型：
--
作者：
Chen K;Tjandra N
通过溶液 NMR 测量的蛋白质主链 15N 自旋弛豫率可提供具有位点特异性分辨率的有用动态信息。传统的方法是记录一系列具有不同弛豫延迟的2D 1H-15N HSQC谱，并从以下共振强度曲线拟合中得出弛豫率。光谱分辨率较差的蛋白质通常需要在 15N/13C 双标记蛋白质样品上收集多个 3D HNCO 光谱。为了减少松弛维数 Carr 等人。 (J. Magn. Reson. (1998) 132, 25–33) 采用手风琴型 HNCO 脉冲序列，通过弛豫干扰无感应衰减 (FID) 数据的数值拟合来获得 15N 或 13C T1 弛豫率。为了避免对时域数据进行密集分析，我们提出了一种修改后的协议，用于从 Accordion-HNCO 谱中轻松获得的线宽中测量 15N T1 和 T2 弛豫率。 T1和T2弛豫可以同时分别卷积成13C'和15N的恒定时间演化周期。弛豫延迟允许达到至少 3×T1 或 3×T2，以便信号在 FID 结束时大幅衰减，所得峰半高全宽 (FWHH) 可直接用于计算弛豫率。当应用于 76 个残基泛素和 226 个残基谷氨酰胺结合蛋白 (GlnBP) 时，该方法产生的 T1 和 T2 值与基于传统 2D 方法的测量结果平均分别偏差 4-6% 和 5-7%。相比之下，传统方法的 T1 固有误差范围为 2-4%，T2 固有误差范围为 3-6%。除了具有可比较的精度外，这里介绍的完全松弛 Accordion HNCO 方法还可以测量 2D 光谱中未解析的共振的松弛率，从而提供更完整的蛋白质动态图像。
Protein backbone 15N spin relaxation rates measured by solution NMR provide useful dynamic information with a site-specific resolution. The conventional method is to record a series of 2D 1H-15N HSQC spectra with varied relaxation delays, and derive relaxation rate from the following curve fitting on the resonance intensities. Proteins with poorly resolved spectra often require several 3D HNCO spectra to be collected on a 15N/13C double labeled protein sample. In order to reduce the relaxation dimension Carr et al. (J. Magn. Reson. (1998) 132, 25–33) employed an Accordion type HNCO pulse sequence to obtain 15N or 13C T1 relaxation rates by numerical fitting of the relaxation interfered free induction decay (FID) data. To avoid intensive analysis of the time domain data, we propose a modified protocol to measure 15N T1 and T2 relaxation rates from easily obtained line-widths in an Accordion-HNCO spectrum. Both T1 and T2 relaxation could be simultaneously convoluted into the constant-time evolution periods of 13C′ and 15N, respectively. The relaxation delay was allowed to reach at least 3×T1 or 3×T2 so that the signal was substantially decayed by the end of the FID, and the resulting peak Full-Width at Half Height (FWHH) could be directly used to calculate relaxation rate. When applied to the 76-residue Ubiquitin and the 226-residue Glutamine-Binding Protein (GlnBP), this method yielded T1 and T2 values deviating on average by 4–6% and 5–7%, respectively, from the measurements based on the conventional 2D method. In comparison, the conventional methods possessed intrinsic error ranges of 2–4% for T1 and 3–6% for T2. In addition to comparable accuracy, the fully-relaxed Accordion HNCO method presented here allowed measurements of relaxation rates for resonances unresolved in 2D spectra, thus providing a more complete dynamic picture of the protein.
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