Highly Selective Stable Isotope Labeling of Histidine Residues by Using a Novel Precursor in <i>E. coli</i>‐Based Overexpression Systems

Schörghuber, Julia; Geist, Leonhard; Platzer, Gerald; Konrat, Robert; Lichtenecker, Roman

doi:10.1002/cbic.201700192

Cited by 10 publications

(11 citation statements)

References 60 publications

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“…Again, the reversible transaminase EC 2.6.1.38 reaction ensured an effective in-vivo conversion to the target residue. In this case we applied the stable enol-tautomer of imidazolepyruvate as a labeling precursor (Schörghuber et al 2017b ). All of the identified precursor compounds mentioned showed highly selective labeling of the corresponding target residues in absence of any cross-labeling to undesired atomic positions.…”

Section: Precursor Identificationmentioning

confidence: 99%

Late metabolic precursors for selective aromatic residue labeling

et al. 2018

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In recent years, we developed a toolbox of heavy isotope containing compounds, which serve as metabolic amino acid precursors in the E. coli-based overexpression of aromatic residue labeled proteins. Our labeling techniques show excellent results both in terms of selectivity and isotope incorporation levels. They are additionally distinguished by low sample production costs and meet the economic demands to further implement protein NMR spectroscopy as a routinely used method in drug development processes. Different isotopologues allow for the assembly of optimized protein samples, which fulfill the requirements of various NMR experiments to elucidate protein structures, analyze conformational dynamics, or probe interaction surfaces. In the present article, we want to summarize the precursors we developed so far and give examples of their special value in the probing of protein–ligand interaction.

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Section: Precursor Identificationmentioning

confidence: 99%

Late metabolic precursors for selective aromatic residue labeling

et al. 2018

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“…Additionally different aromatic sites (Phe and Tyr ε, and Trp ζ3 and ζ2) and α positions (all except Leu) get 13 C labeled and accessible for NMR dynamic studies as well. The other two, using ribose (this work) or precursors closer to the products (Schörghuber et al 2015 , 2017 ) are more discriminating in the positions that get 13 C labeled and can thereby solve potential overlap problems.…”

Section: Resultsmentioning

confidence: 99%

“…No precise values of 13 C incorporation have been reported for the latter approaches (Schörghuber et al 2015 , 2017 ) nor have all positions been targeted (Trp δ1, and His α, β and δ2 are still missing). However this seems relatively straight forward to achieve and could be superior, because the starting compounds are closer to the products.…”

Section: Resultsmentioning

confidence: 99%

“…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 ).…”

Section: Introductionmentioning

confidence: 99%

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

Weininger

2017

J Biomol NMR

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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.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-017-0130-9) contains supplementary material, which is available to authorized users.

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“…On the contrary, anthranilic acid is metabolized to the target amino acid in a well-defined way without losing labeling selectivity at the major metabolic intersection points of the shikimate pathway. Our novel precursor completes our toolbox of isotope labeled compounds to transfer 13 C/ 2 H-patterns onto the aromatic side chains of phenylalanine and tyrosine (Lichtenecker et al 2013b ), tryptophan (Schörghuber et al 2015 ), as well as histidine (Schörghuber et al 2017 ), with every precursor targeting one specific type of residue. This residue selective isotope incorporation cannot be achieved using the early metabolic precursors mentioned above, but is of major importance in the case of high molecular weight protein samples, when signal overlap in the aromatic region becomes a serious issue.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2017

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The application of metabolic precursors for selective stable isotope labeling of aromatic residues in cell-based protein overexpression has already resulted in numerous NMR probes to study the structural and dynamic characteristics of proteins. With anthranilic acid, we present the structurally simplest precursor for exclusive tryptophan side chain labeling. A synthetic route to 13C, 2H isotopologues allows the installation of isolated 13C–1H spin systems in the indole ring of tryptophan, representing a versatile tool to investigate side chain motion using relaxation-based experiments without the loss of magnetization due to strong 1JCC and weaker 2JCH scalar couplings, as well as dipolar interactions with remote hydrogens. In this article, we want to introduce this novel precursor in the context of hitherto existing techniques of in vivo aromatic residue labeling.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-017-0129-2) contains supplementary material, which is available to authorized users.

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Highly Selective Stable Isotope Labeling of Histidine Residues by Using a Novel Precursor in E. coli‐Based Overexpression Systems

Cited by 10 publications

References 60 publications

Late metabolic precursors for selective aromatic residue labeling

Late metabolic precursors for selective aromatic residue labeling

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

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

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