Ramaraju Bhargavi, McFeeters Hana, Vogler Bernhard, McFeeters Robert L
Department of Chemistry, University of Alabama in Huntsville, Huntsville, AL, 35899, USA.
J Biomol NMR. 2017 Jan;67(1):23-34. doi: 10.1007/s10858-016-0081-6. Epub 2016 Dec 27.
Nuclear magnetic resonance spectroscopy studies of ever larger systems have benefited from many different forms of isotope labeling, in particular, site specific isotopic labeling. Site specific C labeling of methyl groups has become an established means of probing systems not amenable to traditional methodology. However useful, methyl reporter sites can be limited in number and/or location. Therefore, new complementary site specific isotope labeling strategies are valuable. Aromatic amino acids make excellent probes since they are often found at important interaction interfaces and play significant structural roles. Aromatic side chains have many of the same advantages as methyl containing amino acids including distinct C chemical shifts and multiple magnetically equivalent H positions. Herein we report economical bacterial production and one-step purification of phenylalanine with C incorporation at the Cα, Cγ and Cε positions, resulting in two isolated H-C spin systems. We also present methodology to maximize incorporation of phenylalanine into recombinantly overexpressed proteins in bacteria and demonstrate compatibility with ILV-methyl labeling. Inexpensive, site specific isotope labeled phenylalanine adds another dimension to biomolecular NMR, opening new avenues of study.
对越来越大的系统进行核磁共振波谱研究受益于多种不同形式的同位素标记，特别是位点特异性同位素标记。甲基基团的位点特异性碳标记已成为探测不适用于传统方法的系统的既定手段。然而，甲基报告位点在数量和/或位置上可能受到限制。因此，新的互补位点特异性同位素标记策略很有价值。芳香族氨基酸是优秀的探针，因为它们经常出现在重要的相互作用界面并发挥重要的结构作用。芳香族侧链具有许多与含甲基氨基酸相同的优点，包括独特的碳化学位移和多个磁等价氢位置。在此，我们报告了在Cα、Cγ和Cε位置掺入碳的苯丙氨酸的经济的细菌生产和一步纯化方法，产生了两个孤立的氢-碳自旋系统。我们还提出了使苯丙氨酸最大程度掺入细菌中重组过表达蛋白质的方法，并证明了与异亮氨酸-亮氨酸-缬氨酸-甲基标记的兼容性。廉价的、位点特异性同位素标记的苯丙氨酸为生物分子核磁共振增添了新的维度，开辟了新的研究途径。
