Weininger Ulrich
Department of Biophysical Chemistry, Center for Molecular Protein Science, Lund University, P. O. Box 124, 22100, Lund, Sweden.
Institute of Physics, Biophysics, Martin-Luther-University Halle-Wittenberg, 06120, Halle (Saale), Germany.
J Biomol NMR. 2017 Sep;69(1):23-30. doi: 10.1007/s10858-017-0130-9. Epub 2017 Aug 30.
Experimental studies on protein dynamics at atomic resolution by NMR-spectroscopy in solution require isolated H-X spin pairs. This is the default scenario in standard H-N backbone experiments. Side chain dynamic experiments, which allow to study specific local processes like proton-transfer, or tautomerization, require isolated H-C sites which must be produced by site-selective C labeling. In the most general way this is achieved by using site-selectively C-enriched glucose as the carbon source in bacterial expression systems. Here we systematically investigate the use of site-selectively C-enriched ribose as a suitable precursor for C labeled histidines and tryptophans. The C incorporation in nearly all sites of all 20 amino acids was quantified and compared to glucose based labeling. In general the ribose approach results in more selective labeling. 1-C ribose exclusively labels His δ2 and Trp δ1 in aromatic side chains and helps to resolve possible overlap problems. The incorporation yield is however only 37% in total and 72% compared to yields of 2-C glucose. A combined approach of 1-C ribose and 2-C glucose maximizes C incorporation to 75% in total and 150% compared to 2-C glucose only. Further histidine positions β, α and CO become significantly labeled at around 50% in total by 3-, 4- or 5-C ribose. Interestingly backbone CO of Gly, Ala, Cys, Ser, Val, Phe and Tyr are labeled at 40-50% in total with 3-C ribose, compared to 5% and below for 1-C and 2-C glucose. Using ribose instead of glucose as a source for site-selective C labeling enables a very selective labeling of certain positions and thereby expanding the toolbox for customized isotope labeling of amino-acids.
通过溶液中的核磁共振光谱在原子分辨率下对蛋白质动力学进行实验研究需要分离的H-X自旋对。这是标准H-N主链实验中的默认情况。侧链动力学实验允许研究特定的局部过程，如质子转移或互变异构，需要分离的H-C位点，这些位点必须通过位点选择性C标记产生。最常见的方法是在细菌表达系统中使用位点选择性富集C的葡萄糖作为碳源。在这里，我们系统地研究了位点选择性富集C的核糖作为C标记组氨酸和色氨酸的合适前体的用途。对所有20种氨基酸几乎所有位点的C掺入进行了定量，并与基于葡萄糖的标记进行了比较。一般来说，核糖方法导致更具选择性的标记。1-C核糖专门标记芳香侧链中的His δ2和Trp δ1，并有助于解决可能的重叠问题。然而，掺入产率总计仅为37%，与2-C葡萄糖的产率相比为72%。1-C核糖和2-C葡萄糖的组合方法使C掺入总量最大化至75%，与仅使用2-C葡萄糖相比为150%。进一步的组氨酸位置β、α和CO通过3-C、4-C或5-C核糖总计约50%被显著标记。有趣的是，Gly、Ala、Cys、Ser、Val、Phe和Tyr的主链CO用3-C核糖总计标记为40-50%，而1-C和2-C葡萄糖的标记率为5%及以下。使用核糖而不是葡萄糖作为位点选择性C标记的来源能够对某些位置进行非常有选择性的标记，从而扩展了用于氨基酸定制同位素标记的工具箱。
