Rosen M K, Gardner K H, Willis R C, Parris W E, Pawson T, Kay L E
Department of Medical Genetics, University of Toronto, Ontario, Canada.
J Mol Biol. 1996 Nov 15;263(5):627-36. doi: 10.1006/jmbi.1996.0603.
Deuteration of aliphatic sites in proteins has shown great potential to increase the range of molecules amenable to study by NMR spectroscopy. One problem inherent in high-level deuterium incorporation is the loss of 1H-1H distance information obtainable from NOESY spectra of the labeled proteins. In the limit of perdeuteration, the available NH-NH NOEs are insufficient in many cases to define the three-dimensional structure of a folded protein. We describe here a method of producing proteins that retains all the advantages of perdeuteration, while enabling observation of many NOEs absent from spectra of fully deuterated samples. Overexpression of proteins in bacteria grown in 2H2O medium containing protonated pyruvate as the sole carbon source results in complete deuteration at C alpha and > 80% deuteration at C beta positions of nearly all amino acids. In contrast, the methyl groups of Ala, Val, Leu and Ile (gamma 2 only) remain highly protonated. This labeling pattern can be readily understood from analysis of bacterial pathways for pyruvate utilization and amino acid biosynthesis. As Ala, Val, Leu and Ile are among the most highly represented residue types in protein hydrophobic cores and at protein-protein interfaces, selectively methyl-protonated samples will be useful in many areas of structural analysis of larger molecules and molecular complexes by NMR.
蛋白质中脂肪族位点的氘代已显示出巨大潜力，可扩大适合通过核磁共振光谱学研究的分子范围。高水平氘掺入所固有的一个问题是，从标记蛋白质的NOESY谱中可获得的1H-1H距离信息会丢失。在全氘代的极限情况下，许多情况下可用的NH-NH NOE不足以确定折叠蛋白质的三维结构。我们在此描述一种生产蛋白质的方法，该方法保留了全氘代的所有优点，同时能够观察到全氘代样品谱中不存在的许多NOE。在含有质子化丙酮酸作为唯一碳源的2H2O培养基中生长的细菌中过量表达蛋白质，几乎所有氨基酸的Cα位置会完全氘代，Cβ位置的氘代率>80%。相比之下，丙氨酸、缬氨酸、亮氨酸和异亮氨酸的甲基（仅γ2）仍高度质子化。通过分析丙酮酸利用和氨基酸生物合成的细菌途径，可以很容易理解这种标记模式。由于丙氨酸、缬氨酸、亮氨酸和异亮氨酸是蛋白质疏水核心和蛋白质-蛋白质界面中最常见的残基类型，选择性甲基质子化的样品将在通过核磁共振对大分子和分子复合物进行结构分析的许多领域中发挥作用。
