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

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