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A set of engineered Escherichia coli expression strains for selective isotope and reactivity labeling of amino acid side chains and flavin cofactors.

Mehlhorn J, Steinocher H, Beck S, Kennis JT, Hegemann P, Mathes T - PLoS ONE (2013)

Bottom Line: For flavin labeling we report optimized auxotrophic strains with significantly enhanced flavin uptake properties.Labeled protein biosynthesis using these strains was achieved in optimized cultivation procedures using high cell density fermentation.Finally, we demonstrate how this approach is used for a clear assignment of vibrational spectroscopic difference signals of apoprotein and cofactor of a flavin containing photoreceptor of the BLUF (Blue Light receptors Using FAD) family.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biologie/Experimentelle Biophysik, Humboldt-Universität zu Berlin, Berlin, Germany.

ABSTRACT
Biological reactions are facilitated by delicate molecular interactions between proteins, cofactors and substrates. To study and understand their dynamic interactions researchers have to take great care not to influence or distort the object of study. As a non-invasive alternative to a site-directed mutagenesis approach, selective isotope labeling in combination with vibrational spectroscopy may be employed to directly identify structural transitions in wild type proteins. Here we present a set of customized Escherichia coli expression strains, suitable for replacing both the flavin cofactor and/or selective amino acids with isotope enriched or chemically modified substrates. For flavin labeling we report optimized auxotrophic strains with significantly enhanced flavin uptake properties. Labeled protein biosynthesis using these strains was achieved in optimized cultivation procedures using high cell density fermentation. Finally, we demonstrate how this approach is used for a clear assignment of vibrational spectroscopic difference signals of apoprotein and cofactor of a flavin containing photoreceptor of the BLUF (Blue Light receptors Using FAD) family.

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Related in: MedlinePlus

Tyrosine requirement of CpXΔY.Cells were grown in minimal medium supplied with the indicated amount of L-tyrosine. The cell density was estimated by monitoring the optical density at 600 nm at the indicated times (A). Cell densities in the stationary phase for two different clones were plotted against the tyrosine concentration (B). Thereby the relative amount of amino acid substrate to glucose for maximum cell density may be determined.
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pone-0079006-g003: Tyrosine requirement of CpXΔY.Cells were grown in minimal medium supplied with the indicated amount of L-tyrosine. The cell density was estimated by monitoring the optical density at 600 nm at the indicated times (A). Cell densities in the stationary phase for two different clones were plotted against the tyrosine concentration (B). Thereby the relative amount of amino acid substrate to glucose for maximum cell density may be determined.

Mentions: In contrast to all other strains described here the glutamine auxotrophic strains (Table 2) showed significant growth limitation on LB plates compared to wild type E. coli (not shown). Accordingly, these strains require high amounts of glutamine relative to the carbon source for optimal growth in minimal medium (Table 2). In M9 medium without amino acid supplementation all strains were unable to grow (Figure 3, Figure S1). While most of the here presented strains are single gene knockouts, the asparagine auxotrophic strain CpXΔN required two genes, asnA and asnB, to be removed for a complete asparagine auxotrophic phenotype. asnB encodes for a glutamine dependent asparagine synthase and may lead to nitrogen isotope dilution through unlabeled de novo synthesized glutamine. Since this enzyme may also lead to isotope scrambling from glutamine into asparagine in the glutamine auxotrophic strain CpXΔQ, we additionally removed asnB in the modified glutamine auxotrophic strain CpXΔQ*. Both glutamine auxotrophic strains however show virtually identically glutamine requirements, which might indicate that this pathway is rather insignificant for nitrogen isotope scrambling between asparagine and glutamine in these strains (Figure S2, Table 2). However, we did not further investigate in this direction here. Since the vitality of the double glnA-, asnB- mutant CpXΔQ* is apparently not affected by the additional mutation, we suggest using CpXΔQ* for further experiments.


A set of engineered Escherichia coli expression strains for selective isotope and reactivity labeling of amino acid side chains and flavin cofactors.

Mehlhorn J, Steinocher H, Beck S, Kennis JT, Hegemann P, Mathes T - PLoS ONE (2013)

Tyrosine requirement of CpXΔY.Cells were grown in minimal medium supplied with the indicated amount of L-tyrosine. The cell density was estimated by monitoring the optical density at 600 nm at the indicated times (A). Cell densities in the stationary phase for two different clones were plotted against the tyrosine concentration (B). Thereby the relative amount of amino acid substrate to glucose for maximum cell density may be determined.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3815312&req=5

pone-0079006-g003: Tyrosine requirement of CpXΔY.Cells were grown in minimal medium supplied with the indicated amount of L-tyrosine. The cell density was estimated by monitoring the optical density at 600 nm at the indicated times (A). Cell densities in the stationary phase for two different clones were plotted against the tyrosine concentration (B). Thereby the relative amount of amino acid substrate to glucose for maximum cell density may be determined.
Mentions: In contrast to all other strains described here the glutamine auxotrophic strains (Table 2) showed significant growth limitation on LB plates compared to wild type E. coli (not shown). Accordingly, these strains require high amounts of glutamine relative to the carbon source for optimal growth in minimal medium (Table 2). In M9 medium without amino acid supplementation all strains were unable to grow (Figure 3, Figure S1). While most of the here presented strains are single gene knockouts, the asparagine auxotrophic strain CpXΔN required two genes, asnA and asnB, to be removed for a complete asparagine auxotrophic phenotype. asnB encodes for a glutamine dependent asparagine synthase and may lead to nitrogen isotope dilution through unlabeled de novo synthesized glutamine. Since this enzyme may also lead to isotope scrambling from glutamine into asparagine in the glutamine auxotrophic strain CpXΔQ, we additionally removed asnB in the modified glutamine auxotrophic strain CpXΔQ*. Both glutamine auxotrophic strains however show virtually identically glutamine requirements, which might indicate that this pathway is rather insignificant for nitrogen isotope scrambling between asparagine and glutamine in these strains (Figure S2, Table 2). However, we did not further investigate in this direction here. Since the vitality of the double glnA-, asnB- mutant CpXΔQ* is apparently not affected by the additional mutation, we suggest using CpXΔQ* for further experiments.

Bottom Line: For flavin labeling we report optimized auxotrophic strains with significantly enhanced flavin uptake properties.Labeled protein biosynthesis using these strains was achieved in optimized cultivation procedures using high cell density fermentation.Finally, we demonstrate how this approach is used for a clear assignment of vibrational spectroscopic difference signals of apoprotein and cofactor of a flavin containing photoreceptor of the BLUF (Blue Light receptors Using FAD) family.

View Article: PubMed Central - PubMed

Affiliation: Institut für Biologie/Experimentelle Biophysik, Humboldt-Universität zu Berlin, Berlin, Germany.

ABSTRACT
Biological reactions are facilitated by delicate molecular interactions between proteins, cofactors and substrates. To study and understand their dynamic interactions researchers have to take great care not to influence or distort the object of study. As a non-invasive alternative to a site-directed mutagenesis approach, selective isotope labeling in combination with vibrational spectroscopy may be employed to directly identify structural transitions in wild type proteins. Here we present a set of customized Escherichia coli expression strains, suitable for replacing both the flavin cofactor and/or selective amino acids with isotope enriched or chemically modified substrates. For flavin labeling we report optimized auxotrophic strains with significantly enhanced flavin uptake properties. Labeled protein biosynthesis using these strains was achieved in optimized cultivation procedures using high cell density fermentation. Finally, we demonstrate how this approach is used for a clear assignment of vibrational spectroscopic difference signals of apoprotein and cofactor of a flavin containing photoreceptor of the BLUF (Blue Light receptors Using FAD) family.

Show MeSH
Related in: MedlinePlus