Applications of REDOR for Distance Measurements in Biological Solids

Grage, Stephan L.; Watts, Anthony

doi:10.1016/s0066-4103(06)60005-7

Cited by 19 publications

(8 citation statements)

References 127 publications

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“…In some cases a single cofactor-specific or amino acid-specific labeling may not be sufficient for a clear assignment and a selective double labeling of cofactor and amino acids is needed. This may be useful for downshifting selected flavin and amino acid signatures simultaneously to visualize underlying signals from other parts of the protein, but also for NMR techniques employing selective heteronuclear coupling to determine distances between flavin and amino acid side chains as well as their mutual orientations [ 53 ]. For this purpose we introduced auxotrophies for selected amino acids into the enhanced riboflavin auxotrophic strain CpXribF (see above).…”

Section: Resultsmentioning

confidence: 99%

A Set of Engineered Escherichia coli Expression Strains for Selective Isotope and Reactivity Labeling of Amino Acid Side Chains and Flavin Cofactors

et al. 2013

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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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Section: Resultsmentioning

confidence: 99%

A Set of Engineered Escherichia coli Expression Strains for Selective Isotope and Reactivity Labeling of Amino Acid Side Chains and Flavin Cofactors

et al. 2013

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“…The 40-ppm carbon was hypothesized to be bonded to nitrogen, based on its chemical shift and the presence of a single nitrogen amine peak in the 15 N CPMAS spectrum of a uniformly 15 N biosynthetically labeled sample for which cells were grown in the presence of 15 NH 4 Cl. This possibility was investigated by 13 C{ 15 N}Rotational-Echo Double Resonance (REDOR), a solid-state NMR experiment that enables the measurement of heteronuclear dipolar couplings between heteronuclear spin pairs in a sample [12, 13, 29]. In this case, a one-bond 13 C{ 15 N} REDOR filter revealed that the 40-ppm carbon was completely dephased and was directly bonded to a nitrogen.…”

Section: Solid-state Nmr Insights Into Bacterial Biofilm Compositionmentioning

confidence: 99%

Solid-state NMR for bacterial biofilms

Reichhardt

Cegelski

2013

Molecular Physics

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Bacteria associate with surfaces and one another by elaborating an extracellular matrix to encapsulate cells, creating communities termed biofilms. Biofilms are beneficial in some ecological niches, but also contribute to the pathogenesis of serious and chronic infectious diseases. New approaches and quantitative measurements are needed to define the composition and architecture of bacterial biofilms to help drive the development of strategies to interfere with biofilm assembly. Solid-state NMR is uniquely suited to the examination of insoluble and complex macromolecular and whole-cell systems. This article highlights three examples that implement solid-state NMR to deliver insights into bacterial biofilm composition and changes in cell-wall composition as cells transition to the biofilm lifestyle. Most recently, solid-state NMR measurements provided a total accounting of the protein and polysaccharide components in the extracellular matrix of an E. coli biofilm and transform our qualitative descriptions of matrix composition into chemical parameters that permit quantitative comparisons among samples. We present additional data for whole biofilm samples (cells plus the extracellular matrix) that complement matrix-only analyses. The study of bacterial biofilms by solid-state NMR is an exciting avenue ripe with many opportunities and we close the article by articulating some outstanding questions and future directions in this area.

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“…A brief primer on the REDOR experiment follows. More complete descriptions of the evolution of the dipolar coupling are beyond the scope of this digest and are available in the references cited here 3, 7, 8 .…”

Section: Redor Access To Dipolar Couplingsmentioning

confidence: 99%

REDOR NMR for drug discovery

Cegelski

2013

Bioorganic & Medicinal Chemistry Letters

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Rotational-Echo DOuble-Resonance (REDOR) NMR is a powerful and versatile solid-state NMR measurement that has been recruited to elucidate drug modes of action and to drive the design of new therapeutics. REDOR has been implemented to examine composition, structure, and dynamics in diverse macromolecular and whole-cell systems, including taxol-bound microtubules, enzyme-cofactor-inhibitor ternary complexes, and antibiotic-whole-cell complexes. The REDOR approach involves the integrated design of specific isotopic labeling strategies and the selection of appropriate REDOR experiments. By way of example, this digest illustrates the versatility of the REDOR approach, with an emphasis on the practical considerations of experimental design and data interpretation.

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Applications of REDOR for Distance Measurements in Biological Solids

Cited by 19 publications

References 127 publications

A Set of Engineered Escherichia coli Expression Strains for Selective Isotope and Reactivity Labeling of Amino Acid Side Chains and Flavin Cofactors

A Set of Engineered Escherichia coli Expression Strains for Selective Isotope and Reactivity Labeling of Amino Acid Side Chains and Flavin Cofactors

Solid-state NMR for bacterial biofilms

REDOR NMR for drug discovery

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