Synthesis of Metabolic Amino acid Precursors: Tools for Selective Isotope Labeling in Cell-Based Protein Overexpression

Lichtenecker, Roman; Schörghuber, Julia; Bisaccia, Marilena

doi:10.1055/s-0034-1378878

Cited by 11 publications

(10 citation statements)

References 26 publications

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“…These compounds have been prepared via multistep organic synthesis, which we optimized in terms of robustness, yields, labeling selectivity and costs (see the corresponding literature for details). We used commercially available sources of 13 C as starting materials or reagents, such as isotopologues of acetone, glycine, potassium cyanide and formaldehyde (Lichtenecker 2014 ; Lichtenecker et al 2015 ). Deuterium patterns have been exclusively derived from deuterium oxide, which is the cheapest source of 2 H available.…”

Section: Precursor Synthesismentioning

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 Synthesismentioning

confidence: 99%

Late metabolic precursors for selective aromatic residue labeling

et al. 2018

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“…All single 15 N labeled amino acids as well as labeled αketoisovaleric acid (3-methyl- 13 C, 3, 4, 4, 4-D4) (KIV) were purchased from Cambridge Isotope Laboratories, Inc. (USA). Labeled 4-methyl-2-oxovaleric acid (4methyl-13 C, 3, 3, 4, 5, 5, 5-D6) (MOV) was synthesized as reported 20 . 6-diazo-5-oxo-L-norleucine (DON), aminooxyacetate (AOA), and D-malate (DM) were obtained from Sigma-Aldrich (USA).…”

Section: Methodsmentioning

confidence: 99%

E. coli “Stablelabel” S30 lysate for optimized cell-free NMR sample preparation

Levin

Löhr

Karakoc

et al. 2023

Preprint

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Cell-free (CF) synthesis with highly productive E. coli lysates is a convenient method to produce labeled proteins for NMR studies. Despite reduced metabolic activity in CF lysates, a certain scrambling of supplied isotope labels is still notable. Most problematic are conversions of 15N labels of the amino acids L-Asp, LAsn, L-Gln, L-Glu and L-Ala, resulting into ambiguous NMR signals as well as into label dilution. Specific inhibitor cocktails could suppress most undesired conversion reactions, while limited availability and potential side effects on CF system productivity need to be considered. As alternative route to address NMR label conversion in CF systems, we describe the generation of optimized E. coli lysates with reduced amino acid scrambling activity. Our strategy is based on the proteome blueprint of standardized CF S30 lysates of the E. coli strain A19. Identified lysate enzymes with suspected amino acid scrambling activity were eliminated by constructing corresponding single and cumulative chromosomal mutations in A19. CF lysates prepared from the mutants were analyzed for their CF protein synthesis efficiency and for residual scrambling activity. The A19 derivative “Stablelabel” containing the cumulative mutations asnA, ansA/B, glnA, aspC and ilvE was finally identified as giving the most useful CF S30 lysates. We demonstrate the optimized NMR spectral complexity of selectively labeled proteins CF synthesized in “Stablelabel” lysates. By taking advantage of the ilvE deletion in "Stablelabel", we further exemplify a new strategy for methyl group specific labeling of membrane proteins with the proton pump proteorhodopsin.

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“…Other labelled biosynthetic precursors such as precursors for labelling the indole in Trp have been explored and are valuable tools for residue-specific isotope labelling. 42 However, metabolic labelling processes used for residue specific labelling might cause potential scrambling of isotope labels due to metabolic interconversion of the amino acids. 43,44 Labelling of single amino acid residues is a potential strategy when focusing on one or very few key positions in a protein.…”

Section: Rsc Chemical Biology Accepted Manuscriptmentioning

confidence: 99%