Site-selective (13)C labeling of histidine and tryptophan using ribose

Experimental studies on protein dynamics at atomic resolution by NMR-spectroscopy in solution require isolated (1)H-X spin pairs. This is the default scenario in standard (1)H-(15)N backbone experiments. Side chain dynamic experiments, which allow to study specific local processes like proton-transfer, or tautomerization, require isolated (1)H-(13)C sites which must be produced by site-selective (13)C labeling. In the most general way this is achieved by using site-selectively (13)C-enriched glucose as the carbon source in bacterial expression systems. Here we systematically investigate the use of site-selectively (13)C-enriched ribose as a suitable precursor for (13)C labeled histidines and tryptophans. The (13)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-(13)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-(13)C glucose. A combined approach of 1-(13)C ribose and 2-(13)C glucose maximizes (13)C incorporation to 75% in total and 150% compared to 2-(13)C glucose only. Further histidine positions β, α and CO become significantly labeled at around 50% in total by 3-, 4- or 5-(13)C ribose. Interestingly backbone CO of Gly, Ala, Cys, Ser, Val, Phe and Tyr are labeled at 40–50% in total with 3-(13)C ribose, compared to 5% and below for 1-(13)C and 2-(13)C glucose. Using ribose instead of glucose as a source for site-selective (13)C labeling enables a very selective labeling of certain positions and thereby expanding the toolbox for customized isotope labeling of amino-acids. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s10858-017-0130-9) contains supplementary material, which is available to authorized users.