一种 13C 标记策略揭示了钙调蛋白中芳香侧链运动的范围。
A 13C labeling strategy reveals a range of aromatic side chain motion in calmodulin.
机构信息
Graduate Group in Biochemistry and Molecular Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA.
出版信息
J Am Chem Soc. 2013 Jul 3;135(26):9560-3. doi: 10.1021/ja4001129. Epub 2013 Jun 19.
Abstract
NMR relaxation experiments often require site-specific isotopic enrichment schemes in order to allow for quantitative interpretation. Here we describe a new labeling scheme for site-specific (13)C-(1)H enrichment of a single ortho position of aromatic amino acid side chains in an otherwise perdeuterated background by employing a combination of [4-(13)C]erythrose and deuterated pyruvate during growth on deuterium oxide. This labeling scheme largely eliminates undesired contributions to (13)C relaxation and greatly simplifies the fitting of relaxation data using the Lipari-Szabo model-free formalism. This approach is illustrated with calcium-saturated vertebrate calmodulin and oxidized flavodoxin from Cyanobacterium anabaena . Analysis of (13)C relaxation in the aromatic groups of calcium-saturated calmodulin indicates a wide range of motion in the subnanosecond time regime.
摘要
NMR 弛豫实验通常需要进行特定部位的同位素富集方案，以便进行定量解释。在这里，我们描述了一种新的标记方案，用于通过在重水生长过程中使用[4-(13)C]赤藓糖和氘代丙酮酸，对芳香族氨基酸侧链的单个邻位进行特定部位（13）C-（1）H 富集，而背景为完全氘代。该标记方案在很大程度上消除了（13）C 弛豫的不需要的贡献，并极大地简化了使用 Lipari-Szabo 无模型形式主义拟合弛豫数据。该方法通过钙饱和脊椎动物钙调蛋白和来自蓝藻的氧化黄素蛋白进行了说明。钙饱和钙调蛋白中芳族基团的（13）C 弛豫分析表明，在亚纳秒时间范围内存在广泛的运动。
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本文引用的文献
1
The functional capacity of the natural amino acids for molecular recognition.天然氨基酸在分子识别方面的功能能力。
Mol Biosyst. 2010 Jul;6(7):1186-94. doi: 10.1039/b927393j. Epub 2010 Apr 9.
2
The role of conformational entropy in molecular recognition by calmodulin.钙调蛋白分子识别中的构象熵作用。
Nat Chem Biol. 2010 May;6(5):352-8. doi: 10.1038/nchembio.347. Epub 2010 Apr 11.
3
SAIL--stereo-array isotope labeling.SAIL--立体阵列同位素标记。
Q Rev Biophys. 2009 Nov;42(4):247-300. doi: 10.1017/S0033583510000016. Epub 2010 Apr 7.
4
Site-specific backbone amide (15)N chemical shift anisotropy tensors in a small protein from liquid crystal and cross-correlated relaxation measurements.液晶和交叉相关弛豫测量中小蛋白中特定位置酰胺（15）N 化学位移各向异性张量。
J Am Chem Soc. 2010 Mar 31;132(12):4295-309. doi: 10.1021/ja910186u.
5
Monitoring aromatic picosecond to nanosecond dynamics in proteins via 13C relaxation: expanding perturbation mapping of the rigidifying core mutation, V54A, in eglin c.通过13C弛豫监测蛋白质中芳香族皮秒到纳秒级动力学：扩展对埃格林c中刚性化核心突变V54A的微扰图谱分析。
Biochemistry. 2008 Apr 29;47(17):4876-86. doi: 10.1021/bi702330t. Epub 2008 Apr 5.
6
Fractional 13C enrichment of isolated carbons using [1-13C]- or [2- 13C]-glucose facilitates the accurate measurement of dynamics at backbone Calpha and side-chain methyl positions in proteins.使用[1-¹³C]-或[2-¹³C]-葡萄糖对分离出的碳进行¹³C富集分数，有助于准确测量蛋白质主链α-碳和侧链甲基位置的动力学。
J Biomol NMR. 2007 Jul;38(3):199-212. doi: 10.1007/s10858-007-9158-6. Epub 2007 Jun 7.
7
Characterization of the fast dynamics of protein amino acid side chains using NMR relaxation in solution.利用溶液中的核磁共振弛豫表征蛋白质氨基酸侧链的快速动力学。
Chem Rev. 2006 May;106(5):1672-99. doi: 10.1021/cr040422h.
8
Fast time scale dynamics of protein backbones: NMR relaxation methods, applications, and functional consequences.蛋白质主链的快速时间尺度动力学：核磁共振弛豫方法、应用及功能影响
Chem Rev. 2006 May;106(5):1624-71. doi: 10.1021/cr040421p.
9
New tools provide new insights in NMR studies of protein dynamics.新工具为蛋白质动力学的核磁共振研究提供了新见解。
Science. 2006 Apr 14;312(5771):224-8. doi: 10.1126/science.1124964.
10
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