Site-selective 13C labeling of proteins using erythrose

Abstract

NMR-spectroscopy enables unique experimental studies on protein dynamics at atomic resolution. In order to obtain a full atom view on protein dynamics, and to study specific local processes like ring-flips, proton-transfer, or tautomerization, one has to perform studies on amino-acid side chains. A key requirement for these studies is site-selective labeling with 13C and/or 1H, which is achieved in the most general way by using site-selectively 13C-enriched glucose (1- and 2-13C) as the carbon source in bacterial expression systems. Using this strategy, multiple sites in side chains, including aromatics, become site-selectively labeled and suitable for relaxation studies. Here we systematically investigate the use of site-selectively 13C-enriched erythrose (1-, 2-, 3- and 4-13C) as a suitable precursor for 13C labeled aromatic side chains. We quantify 13C incorporation in nearly all sites in all 20 amino acids and compare the results to glucose based labeling. In general the erythrose approach results in more selective labeling. While there is only a minor gain for phenylalanine and tyrosine side-chains, the 13C incorporation level for tryptophan is at least doubled. Additionally, the Phe ζ and Trp η2 positions become labeled. In the aliphatic side chains, labeling using erythrose yields isolated 13C labels for certain positions, like Ile β and His β, making these sites suitable for dynamics studies. Using erythrose instead of glucose as a source for site-selective 13C labeling enables unique or superior labeling for certain positions and is thereby expanding the toolbox for customized isotope labeling of amino-acid side-chains.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-017-0096-7) contains supplementary material, which is available to authorized users.

Synthesis and incorporation of 13C-labeled DNA building blocks to probe structural dynamics of DNA by NMR

“…A crucial prerequisite to make use of these relaxation-based NMR experiments is the introduction of stable isotopes, such as 13 C, 15 N or 2 H ( 11 , 12 ). For proteins labeling protocols are very advanced and in combination with sophisticated pulse sequences large proteins and even high molecular weight protein complexes can be studied via NMR spectroscopy and methyl group specific 13 C-labeling ( 13 – 19 ). In the field of nucleic acid NMR spectroscopy, however, the isotope labeling strategies relying on the enzymatic incorporation of stable isotope labeled (deoxy)ribonucleotides were established in the mid-90s with minor progress since then ( 20 – 31 ).…”

Anthranilic acid, the new player in the ensemble of aromatic residue labeling precursor compounds

“…Our novel labeling technique represents an advanced development of hitherto applied methods using early metabolic intermediates as heavy isotope sources. Recently, isotopologues of erythrose have been introduced to direct 13 C labeling into defined atomic positions of the aromatic side-chains in phenylalanine, tyrosine and tryptophan (Kasinath et al 2015 ; Weininger 2017 ). In the corresponding protocols, limited isotope incorporation levels in the range of 50–70% for tryptophan, as well as partial isotope scrambling to undesired atomic positions in the aromatic rings have been reported.…”

“…This is in sharp contrast to the current average prizes of commercial suppliers for isotope labeled early metabolic precursors like [1- 13 C]glucose (€240/g) [2- 13 C]glucose (€630/g), [4- 13 C]erythrose (€1100/g), [2- 13 C]pyruvate (€820/g), [3- 13 C]pyruvate (€1200/g) and [2- 13 C]glycerol (€520/g). Considering the high literature-reported concentrations required (1–3 g labeled compound/L medium) to achieve maximum incorporation levels, these cost issues severely affect their application for routine labeling of multiple protein samples (Teilum et al 2006 ; Lundstrom et al 2007 ; Kasinath et al 2013 ; Weininger 2017 ; Milbradt et al 2015 ).…”

“…Scheme 1 summarizes the main metabolites, which have been used to overexpress proteins containing labeled phenylalanine, tyrosine or tryptophan residues. Certain 13 C-patterns of D-glucose (Teilum et al 2006 ; Lundström et al 2007 ), acetate (Wand et al 1995 ), erythrose (Kasinath et al 2013 ; Weininger 2017 ), pyruvate (Guo et al 2009 ; Milbradt et al 2015 ), or glycerol (LeMaster and Kushlan 1996 ; Ahlner et al 2015 ) have been applied as carbon sources to avoid direct 13 C– 13 C coupling in the target residues. These compounds are commercially available and additional organic synthesis is usually not required.…”

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

“…A well established case is the exclusive site selective labeling of methyl groups at high yields which results in superb NMR probes (Ruschak and Kay 2010 ; Tugarinov et al 2006 ; Tugarinov and Kay 2005 ; Weininger et al 2012b ). Aromatic side chains can be targeted specifically by erythrose labeling (Kasinath et al 2013 ; Weininger 2017 ) and more advanced chemically synthesized precursors for labeling of Trp (Schörghuber et al 2015 ), Tyr and Phe (Lichtenecker et al 2013 ) and most recently for His (Schörghuber et al 2017 ). Also advanced in-vitro strategies using the SAIL approach have been developed for Trp (Miyanoiri et al 2011 ), Tyr and Phe (Takeda et al 2010 ).…”