Inverse or direct detect experiments and probes: Which are “best” for in-vivo NMR research of 13C enriched organisms?

Bastawrous, Monica; Tabatabaei-Anaraki, Maryam; Soong, Ronald; Bermel, Wolfgang; Gundy, Marcel; Boenisch, Holger; Heumann, Hermann; Simpson, André J.

doi:10.1016/j.aca.2020.09.065

Cited by 19 publications

(51 citation statements)

References 68 publications

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“…These might also permit to use 13 C-direct detection, which appears to be necessary to characterize disordered proteins at physiological temperatures, , together with 1 Hα- 13 Cα correlations. − Low-γ nuclei detection may actually be adapted to the slower tumbling times observed in cells, notably in absence of uniform deuteration. It may compete with 1 H-detection at GHz fields, ,,,− but this remains to be tested to a large extent.…”

Section: Integrating In-cell Nmr In the Field Of In-cell Structural B...mentioning

confidence: 99%

In-Cell Structural Biology by NMR: The Benefits of the Atomic Scale

Theillet

2022

Chem. Rev.

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In-cell structural biology aims at extracting structural information about proteins or nucleic acids in their native, cellular environment. This emerging field holds great promises and is already providing new facts and outlooks of interest at both fundamental and applied levels. NMR spectroscopy has important contributions on this stage: it brings information on a broad variety of nuclei at the atomic scale, which ensures its great versatility and uniqueness. Here, we detail the methods, the fundamental knowledge and the applications in biomedical engineering related to in-cell NMR. We finally propose a brief overview of the main other techniques in the field (EPR, smFRET, cryo-ET…) to draw some advisable developments for in-cell NMR. In the era of large-scale screenings and deep learning, both accurate and qualitative experimental evidences are as essential as ever to understand the interior life of cells. In-cell structural biology by NMR spectroscopy can generate such a knowledge, and it does so at the atomic scale. This review is meant to deliver comprehensive but accessible information, with advanced technical details and reflections on the methods, the nature of the results and the future of the field. CONTENTS 5.1.1. In-cell EPR 5.1.2. In-cell FRET microscopy 5.1.3. Mass-spectrometry 5.1.4. Cryo-ET 5.2. The specific benefits of in-cell NMR 5.3. The future technical challenges of in-cell NMR 6. Conclusion Author Information

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Section: Integrating In-cell Nmr In the Field Of In-cell Structural B...mentioning

confidence: 99%

In-Cell Structural Biology by NMR: The Benefits of the Atomic Scale

Theillet

2022

Chem. Rev.

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“…In a more challenging example, Figure 4D–F follows anoxic stress inside living Daphnia magna . Oxidative stress is an important consideration in Daphnia magna as they live in bodies of water that go through seasonal (and depth related) oxygen deficiencies, to which they adapt metabolic responses to survive the harsh conditions [40–42] …”

Section: Resultsmentioning

confidence: 99%

“…Oxidative stress is an important consideration in Daphnia magna as they live in bodies of water that go through seasonal (and depth related) oxygen deficiencies, to which they adapt metabolic responses to survive the harsh conditions. [40][41][42] In this example, living Daphnia magna are maintained inside the NMR using a flow system. As the organisms are alive, their intact bodies lead to magnetic susceptibility distortions and broad lineshape, while large signals from lipids mask metabolic information, [43] making it impossible to perform metabolic analysis using standard 1 H NMR (see Figure 4D top spectrum).…”

Section: Monitoring Processesmentioning

confidence: 99%

DREAMTIME NMR Spectroscopy: Targeted Multi‐Compound Selection with Improved Detection Limits

et al. 2022

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NMR/MRI are critical tools for studying molecular structure and interactions but suffer from relatively low sensitivity and spectral overlap. Here, a Nuclear Magnetic Resonance (NMR) approach, termed DREAMTIME, is introduced that provides “a molecular window” inside complex systems, capable of showing only what the user desires, with complete molecular specificity. The user chooses a list of molecules of interest, and the approach detects only those targets while all other molecules are invisible. The approach is demonstrated in whole human blood and urine, small living aquatic organisms in 1D/2D NMR, and MRI. Finally, as proof‐of‐concept, once overlap is removed via DREAMTIME, a novel “multi‐focusing” approach can be used to increase sensitivity. In human blood and urine, sensitivity increases of 7–12 fold over standard 1H NMR are observed. Applicable even to unknowns, DREAMTIME has widespread application, from monitoring product formation in organic chemistry to monitoring/identifying suites of molecular targets in complex media or in vivo.

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“…Indeed, HETCOR has recently been shown to provide increased resolution and reduced contributions from water in 13 C enriched in vivo samples, ultimately allowing the assignment of a similar number of metabolites as HSQC. [48] Figure 2c shows standard HETCOR experiments acquired using hard pulses on the carbon channel. To investigate whether poor inversion reduces signal in the HETCOR, these pulses were substituted for a Broadband Inversion Pulse (BIP).…”

Section: Resultsmentioning

confidence: 99%

Evaluation of double‐tuned single‐sided planar microcoils for the analysis of small ¹³C enriched biological samples using ¹H‐¹³C 2D heteronuclear correlation NMR spectroscopy

Moxley-Paquette

Lane

et al. 2021

Magnetic Reson in Chemistry

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Microcoils provide a cost-effective approach to improve detection limits for mass-limited samples. Single-sided planar microcoils are advantageous in comparison to volume coils, in that the sample can simply be placed on top. However, the considerable drawback is that the RF field that is produced by the coil decreases with distance from the coil surface, which potentially limits more complex multi-pulse NMR pulse sequences. Unfortunately, 1 H NMR alone is not very informative for intact biological samples due to line broadening caused by magnetic susceptibility distortions, and 1 H-13 C 2D NMR correlations are required to provide the additional spectral dispersion for metabolic assignments in vivo or in situ. To our knowledge, double-tuned single-sided microcoils have not been applied for the 2D 1 H-13 C analysis of intact 13 C enriched biological samples. Questions include the following: Can 1 H-13 C 2D NMR be performed on single-sided planar microcoils? If so, do they still hold sensitivity advantages over conventional 5 mm NMR technology for mass limited samples? Here, 2D 1 H-13 C HSQC, HMQC, and HETCOR variants were compared and then applied to 13 C enriched broccoli seeds and Daphnia magna (water fleas). Compared to 5 mm NMR probes, the microcoils showed a sixfold improvement in mass sensitivity (albeit only for a small localized region) and allowed for the identification of metabolites in a single intact D. magna for the first time. Single-sided planar microcoils show practical benefit for 1 H-13 C NMR of intact biological samples, if localized information within $0.7 mm of the 1 mm I.D. planar microcoil surface is of specific interest.

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Inverse or direct detect experiments and probes: Which are “best” for in-vivo NMR research of 13C enriched organisms?

Cited by 19 publications

References 68 publications

In-Cell Structural Biology by NMR: The Benefits of the Atomic Scale

In-Cell Structural Biology by NMR: The Benefits of the Atomic Scale

DREAMTIME NMR Spectroscopy: Targeted Multi‐Compound Selection with Improved Detection Limits

Evaluation of double‐tuned single‐sided planar microcoils for the analysis of small ¹³C enriched biological samples using ¹H‐¹³C 2D heteronuclear correlation NMR spectroscopy

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Inverse or direct detect experiments and probes: Which are “best” for in-vivo NMR research of 13C enriched organisms?

Cited by 19 publications

References 68 publications

In-Cell Structural Biology by NMR: The Benefits of the Atomic Scale

In-Cell Structural Biology by NMR: The Benefits of the Atomic Scale

DREAMTIME NMR Spectroscopy: Targeted Multi‐Compound Selection with Improved Detection Limits

Evaluation of double‐tuned single‐sided planar microcoils for the analysis of small 13C enriched biological samples using 1H‐13C 2D heteronuclear correlation NMR spectroscopy

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Evaluation of double‐tuned single‐sided planar microcoils for the analysis of small ¹³C enriched biological samples using ¹H‐¹³C 2D heteronuclear correlation NMR spectroscopy