Long-range19F15N distance measurements in highly-13C,15N-enriched solid proteins with19F-dephased REDOR shift (FRESH) spectroscopy

Graesser, Daniel T.; Wylie, Benjamin J.; Nieuwkoop, Andrew J.; Franks, W. Trent; Rienstra, Chad M.

doi:10.1002/mrc.2126

Cited by 22 publications

(26 citation statements)

References 32 publications

(64 reference statements)

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“…This technique is a well-established tool in structural biology, and can measure distances with a resolution that rivals X-ray crystallography. (40) The shortest interdimer distance between 13 C- and 19 F-containing residues is remarkably short in the trimer-of-dimers structure (5.6 Å, see Figure 2B) and substantially longer in the hedgerow structure (8.0 Å). In both cases the interdimer distances are shorter than the shortest likely 13 C to 19 F distance within a dimer (8.8 Å in 1qu7), so the REDOR dephasing will be dominated by the interdimer interaction.…”

Section: Resultsmentioning

confidence: 89%

Kinase-active Signaling Complexes of Bacterial Chemoreceptors Do Not Contain Proposed Receptor−Receptor Contacts Observed in Crystal Structures

2010

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The receptor dimers that mediate bacterial chemotaxis form high-order signaling complexes with CheW and the kinase CheA. From the packing arrangement in two crystal structures of different receptor cytoplasmic fragments, two different models have been proposed for receptor signaling arrays: the trimers-of-dimers and hedgerow models. Here we identified an inter-dimer distance that differs substantially in the two models, labeled the atoms defining this distance through isotopic enrichment, and measured it with 19F-13C REDOR. This was done in two types of receptor samples: isolated bacterial membranes containing overexpressed, intact receptor and soluble receptor fragments reconstituted into kinase-active signaling complexes. In both cases, the distance found was not compatible with the receptor dimer-dimer contacts observed in the trimers-of-dimers or in the hedgerow models. Comparisons of simulated and observed REDOR dephasing were used to deduce a closest-approach distance at this interface, which provides a constraint for the possible arrangements of receptor assemblies.

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Section: Resultsmentioning

confidence: 89%

Kinase-active Signaling Complexes of Bacterial Chemoreceptors Do Not Contain Proposed Receptor−Receptor Contacts Observed in Crystal Structures

2010

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“…Although the simplest way to overcome these complications is to simplify the experimental system to an isolated spin pair, either by sparse/specific isotopic labeling [66–68], dilution of labeled molecules in natural abundance [37], or specific incorporation of fluorinated probes either by attaching through a disulfide linkage [69] or biosynthetically as a non-natural amino acid [70], we were motivated to identify an approach applicable to 31 P-dephased REDOR of crystalline compounds, for which these strategies are not an option. Several studies have reported treatments of REDOR dephasing for multispin systems [71–74].…”

Section: Resultsmentioning

confidence: 99%

31 P-dephased, 13 C-detected REDOR for NMR crystallography at natural isotopic abundance

Greenwood

Clay

Rienstra

2017

Journal of Magnetic Resonance

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Typically, the process of NMR-based structure determination relies on accurately measuring a large number of internuclear distances to serve as restraints for simulated annealing calculations. In solids, the rotational-echo double-resonance (REDOR) experiment is a widely used approach to determine heteronuclear dipolar couplings corresponding to distances usually in the range of 1.5–8 Å. A challenge in the interpretation of REDOR data is the degeneracy of symmetric subunits in an oligomer or equivalent molecules in a crystal lattice, which produce REDOR trajectories that depend explicitly on two or more distances instead of one. This degeneracy cannot be overcome by either spin dilution (for molecules containing 31P, 19F and other highly abundant nuclei) or selective pulses (in the case where there is chemical shift degeneracy). For small, crystalline molecules, such as phosphoserine, we demonstrate that as many as five inter-molecular distances must be considered to model 31P-dephased REDOR data accurately. We report excellent agreement between simulation and experiment once lattice couplings, 31P chemical shift anisotropy, and radiofrequency field inhomogeneity are all taken into account. We also discuss the systematic inaccuracies that may result from approximations that consider only the initial slope of the REDOR trajectory and/or that utilize a two- or three-spin system. Furthermore, we demonstrate the applicability of 31P-dephased REDOR for validation or refinement of candidate crystal structures and show that this approach is especially informative for NMR crystallography of 31P-containing molecules.

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“…57-59 For example, distances between distinct 13 C atoms could be probed within the organic phase in enriched bone using standard DARR (Dipolar Assisted Rotational Resonance) and/or PDSD experiments (Proton Driven Spin Diffusion), 60,61 whereas distances between the organic and mineral components could potentially be measured using derivatives of the REDOR and TEDOR (Transferred Echo Double Resonance) experiments that can detect weak couplings in the presence of strong couplings, and can even use the strong couplings for enhanced spectral resolution. 62,63 Even at ∼20% 13 C enrichment, a ∼20 fold increase in NMR signal intensity would be realized in a 1D 13 C spectrum (leading to ∼400 fold decrease in experimental time for equivalent signal to noise ratio) relative to a sample at natural abundance, and provide adequate spin densities to enable most advanced multidimensional experimental techniques.…”

Section: Perspective: Study Of Low-abundance Amino Acids In Bonementioning

confidence: 99%

NMR Investigation of the Role of Osteocalcin and Osteopontin at the Organic–Inorganic Interface in Bone

et al. 2013

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Mechanical resilience of bone tissue decreases with age. The ability to comprehensively probe and understand bone properties could help alleviate this problem. One important aspect of bone quality which has recently been made evident is the presence of dilatational bands formed by osteocalcin (OC) and osteopontin (OPN), which contribute to fracture toughness. However, experimental evidence of the structural role of these two proteins at the organic-mineral interface in bone is still needed. Solid state nuclear magnetic resonance (SSNMR) is emerging as a useful technique in probing molecular level aspects of bone. Here, we present the first SSNMR study of bone tissue from genetically modified mice lacking OC and/or OPN. Probing the mineral phase, the organic matrix and their interface revealed that despite the absence of OC and OPN, the organic matrix and mineral were well preserved, and the overall exposure of collagen to hydroxyapatite (HA) nanoparticles was hardly affected. However, the proximity to HA surface was slightly increased for a number bone components including less abundant amino acids like lysine, suggesting that this is how the tissue compensates for the lack of OC and OPN. Taken together, the NMR data supports the recently proposed model, in which the contribution of OC - OPN to fracture toughness is related to their presence at the extrafibrillar organic-mineral interfaces, where they reinforce the network of mineralized fibrils and form dilatational bands. In effort towards understanding further the structural role of individual amino acids of low abundance in bone, we then explored the possibility of specific 13C enrichment of mouse bone, and report the first SSNMR spectra of 97% 13C lysine-enriched tissues. Results show that such isotopic enrichment allows valuable molecular-level structural information to be extracted, and sheds light on post-translational modifications undergone by specific amino acids in vivo.

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Long-range19F15N distance measurements in highly-13C,15N-enriched solid proteins with19F-dephased REDOR shift (FRESH) spectroscopy

Cited by 22 publications

References 32 publications

Kinase-active Signaling Complexes of Bacterial Chemoreceptors Do Not Contain Proposed Receptor−Receptor Contacts Observed in Crystal Structures

Kinase-active Signaling Complexes of Bacterial Chemoreceptors Do Not Contain Proposed Receptor−Receptor Contacts Observed in Crystal Structures

31 P-dephased, 13 C-detected REDOR for NMR crystallography at natural isotopic abundance

NMR Investigation of the Role of Osteocalcin and Osteopontin at the Organic–Inorganic Interface in Bone

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