Dynamic Nuclear Polarization of Biomembrane Assemblies

Tran, Nhi; Mentink-Vigier, Frédéric; Long, Joanna

doi:10.3390/biom10091246

Cited by 17 publications

(13 citation statements)

References 115 publications

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“…This forms homogeneous MLVs which are then pelleted by ultracentrifugation. Following this, PA and cryoprotectant are added, the sample is subjected to additional freeze–thaw cycles (×15), and finally the MLVs are directly centrifuged into the MAS rotor …”

Section: Sample Constitution For Biomolecular Dnpmentioning

confidence: 99%

Dynamic Nuclear Polarization for Sensitivity Enhancement in Biomolecular Solid-State NMR

et al. 2022

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Solid-state NMR with magic-angle spinning (MAS) is an important method in structural biology. While NMR can provide invaluable information about local geometry on an atomic scale even for large biomolecular assemblies lacking long-range order, it is often limited by low sensitivity due to small nuclear spin polarization in thermal equilibrium. Dynamic nuclear polarization (DNP) has evolved during the last decades to become a powerful method capable of increasing this sensitivity by two to three orders of magnitude, thereby reducing the valuable experimental time from weeks or months to just hours or days; in many cases, this allows experiments that would be otherwise completely unfeasible. In this review, we give an overview of the developments that have opened the field for DNP-enhanced biomolecular solid-state NMR including state-of-the-art applications at fast MAS and high magnetic field. We present DNP mechanisms, polarizing agents, and sample constitution methods suitable for biomolecules. A wide field of biomolecular NMR applications is covered including membrane proteins, amyloid fibrils, large biomolecular assemblies, and biomaterials. Finally, we present perspectives and recent developments that may shape the field of biomolecular DNP in the future.

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Section: Sample Constitution For Biomolecular Dnpmentioning

confidence: 99%

Dynamic Nuclear Polarization for Sensitivity Enhancement in Biomolecular Solid-State NMR

et al. 2022

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“…Magic angle spinning (MAS) solid-state NMR is not limited by molecular correlation times and is particularly useful to study large protein complexes, amyloid fibrils, and membrane proteins. − With the sensitivity gains conferred by dynamic nuclear polarization (DNP), MAS NMR has the sensitivity to detect proteins at their endogenous concentrations in complex biological environments. ,,,− However, the effectiveness of DNP-enhanced MAS NMR is critically dependent on sample composition − and experimental conditions. ,− DNP increases the sensitivity of NMR spectroscopy through the transfer of the large spin polarization of an unpaired electron to nearby nuclei which are typically introduced into a sample by doping with millimolar concentrations of stable biological radicals. − In addition to millimolar concentrations of polarizing agents, a typical DNP sample of a hydrated biomolecule is cryoprotected by the addition of 60% d 8 -glycerol to aid in the formation of vitreous ice, deuterated to ∼90% to aid spin diffusion, frozen to near liquid nitrogen temperatures, and subjected to magic angle spinning. Pioneering work applying DNP MAS NMR to cultured mammalian cells suggested that measurement of low concentrations of proteins inside mammalian cells is possible, but uncertainty about the biological integrity of the cellular sample limits the utility of the structural information for in-cell experiments.…”

Section: Introductionmentioning

confidence: 99%

In-Cell Sensitivity-Enhanced NMR of Intact Viable Mammalian Cells

et al. 2021

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NMR has the resolution and specificity to determine atomic-level protein structures of isotopically labeled proteins in complex environments, and with the sensitivity gains conferred by dynamic nuclear polarization (DNP), NMR has the sensitivity to detect proteins at their endogenous concentrations. However, DNP sensitivity enhancements are critically dependent on experimental conditions and sample composition. While some of these conditions are theoretically compatible with cellular viability, the effects of others on cellular sample integrity are unknown. Uncertainty about the integrity of cellular samples limits the utility of experimental outputs of in-cell experiments. Using several measures, we establish conditions that support DNP enhancements that can enable detection of micromolar concentrations of proteins in experimentally tractable times that are compatible with cellular viability. Taken together, we establish DNP-assisted MAS NMR as a technique for structural investigations of biomolecules in intact viable cells that can be phenotyped both before and after NMR experiments.

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“…Solid-state NMR (SSNMR) is essential to the structural and functional characterization of membrane proteins (MPs) ( Schubeis et al, 2018 ; Radoicic et al, 2014 ; Wylie et al, 2016 ; Mandala et al, 2018 ; Tran et al, 2020 ). SSNMR can study MPs in native or native-like environments, allowing site-specific analysis of protein structure and activity.…”

Section: Introductionmentioning

confidence: 99%

Water Accessibility Refinement of the Extended Structure of KirBac1.1 in the Closed State

Amani

Schwieters

Borcik

et al. 2021

Front. Mol. Biosci.

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NMR structures of membrane proteins are often hampered by poor chemical shift dispersion and internal dynamics which limit resolved distance restraints. However, the ordering and topology of these systems can be defined with site-specific water or lipid proximity. Membrane protein water accessibility surface area is often investigated as a topological function via solid-state NMR. Here we leverage water-edited solid-state NMR measurements in simulated annealing calculations to refine a membrane protein structure. This is demonstrated on the inward rectifier K+ channel KirBac1.1 found in Burkholderia pseudomallei. KirBac1.1 is homologous to human Kir channels, sharing a nearly identical fold. Like many existing Kir channel crystal structures, the 1p7b crystal structure is incomplete, missing 85 out of 333 residues, including the N-terminus and C-terminus. We measure solid-state NMR water proximity information and use this for refinement of KirBac1.1 using the Xplor-NIH structure determination program. Along with predicted dihedral angles and sparse intra- and inter-subunit distances, we refined the residues 1–300 to atomic resolution. All structural quality metrics indicate these restraints are a powerful way forward to solve high quality structures of membrane proteins using NMR.

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Dynamic Nuclear Polarization of Biomembrane Assemblies

Cited by 17 publications

References 115 publications

Dynamic Nuclear Polarization for Sensitivity Enhancement in Biomolecular Solid-State NMR

Dynamic Nuclear Polarization for Sensitivity Enhancement in Biomolecular Solid-State NMR

In-Cell Sensitivity-Enhanced NMR of Intact Viable Mammalian Cells

Water Accessibility Refinement of the Extended Structure of KirBac1.1 in the Closed State

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