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Rapid Quantitative Measurements of Paramagnetic Relaxation Enhancements in Cu(II)-Tagged Proteins by Proton-Detected Solid-State NMR Spectroscopy
The Journal of Physical Chemistry Letters ( IF 4.8 ) Pub Date : 2017-11-20 00:00:00 , DOI: 10.1021/acs.jpclett.7b02709
Dwaipayan Mukhopadhyay 1 , Philippe S. Nadaud 1 , Matthew D. Shannon 1 , Christopher P. Jaroniec 1
Affiliation  

We demonstrate rapid quantitative measurements of site-resolved paramagnetic relaxation enhancements (PREs), which are a source of valuable structural restraints corresponding to electron–nucleus distances in the ∼10–20 Å regime, in solid-state nuclear magnetic resonance (NMR) spectra of proteins containing covalent Cu2+-binding tags. Specifically, using protein GB1 K28C-EDTA-Cu2+ mutant as a model, we show the determination of backbone amide 15N longitudinal and 1H transverse PREs within a few hours of experiment time based on proton-detected 2D or 3D correlation spectra recorded with magic-angle spinning frequencies ≥ ∼ 60 kHz for samples containing ∼10–50 nanomoles of 2H,13C,15N-labeled protein back-exchanged in H2O. Additionally, we show that the electron relaxation time for the Cu2+ center, needed to convert PREs into distances, can be estimated directly from the experimental data. Altogether, these results are important for establishing solid-state NMR based on paramagnetic-tagging as a routine tool for structure determination of natively diamagnetic proteins.

中文翻译:

质子检测的固态NMR光谱对Cu(II)标记的蛋白质中顺磁弛豫增强的快速定量测量

我们展示了快速定量测量的定点顺磁性弛豫增强(PREs),它是固态核磁共振(NMR)光谱中宝贵的结构约束源,其对应于〜10-20Å范围内的电子-核距离。含有共价Cu 2+结合标签的蛋白质。具体而言,使用蛋白质GB1 K28C-EDTA-Cu 2+突变体作为模型,我们显示了基于记录的质子检测到的2D或3D相关光谱,在实验时间的几个小时内测定骨架酰胺15 N纵向和1 H横向PREs对于包含约10–50纳摩尔2 H,13 C的样品,魔角旋转频率≥〜60 kHz ,15 N标记的蛋白质在H 2 O中进行了反向交换。此外,我们表明,可以直接从实验数据中估算将PRE转换为距离所需的Cu 2+中心的电子弛豫时间。总之,这些结果对于建立基于顺磁性标记的固态NMR(作为确定天然抗磁性蛋白质结构的常规工具)非常重要。
更新日期:2017-11-20
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