Graduate Group in Biochemistry and Molecular Biophysics, University of Pennsylvania Perelman School of Medicine, Philadelphia, Pennsylvania 19104, USA.
J Am Chem Soc. 2013 Jul 3;135(26):9560-3. doi: 10.1021/ja4001129. Epub 2013 Jun 19.
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.
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。
