Solid‐State NMR Spectroscopy on Cellular Preparations Enhanced by Dynamic Nuclear Polarization

Renault, Marie; Pawsey, Shane; Bos, Martine P.; Koers, Eline J.; Nand, Deepak; Boxtel, Ria Tommassen-van; Rosay, Mélanie; Tommassen, Jan; Maas, Werner E.; Baldus, Marc

doi:10.1002/anie.201105984

Cited by 170 publications

(143 citation statements)

References 24 publications

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“…2A). In addition, WC preparations can be readily combined with state-of-the-art ssNMR signal enhancement methods operating at low temperature, thereby reducing acquisition times significantly (25). Such technologies are likely to reduce the expression levels needed to perform cellular ssNMR experiments.…”

Section: Discussionmentioning

confidence: 99%

Cellular solid-state nuclear magnetic resonance spectroscopy

Renault¹,

Boxtel²,

Bos³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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180

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Decrypting the structure, function, and molecular interactions of complex molecular machines in their cellular context and at atomic resolution is of prime importance for understanding fundamental physiological processes. Nuclear magnetic resonance is a wellestablished imaging method that can visualize cellular entities at the micrometer scale and can be used to obtain 3D atomic structures under in vitro conditions. Here, we introduce a solid-state NMR approach that provides atomic level insights into cell-associated molecular components. By combining dedicated protein production and labeling schemes with tailored solid-state NMR pulse methods, we obtained structural information of a recombinant integral membrane protein and the major endogenous molecular components in a bacterial environment. Our approach permits studying entire cellular compartments as well as cell-associated proteins at the same time and at atomic resolution. cellular envelope | Escherichia coli | lipoprotein | PagL | magic angle spinning P hysiological processes rely on the concerted action of molecular entities in and across different cellular compartments. Whereas advancements in molecular imaging have provided unprecedented insights into the macromolecular organization in the subnanometer range (1), studying atomic structure and motion in situ has been challenging for structural biology. NMR has provided insight into cellular processes (2-4) and can determine entire 3D molecular structures inside living cells (5) provided that molecular entities tumble rapidly in a cellular setting. In principle, solid-state NMR (ssNMR) spectroscopy offers a complementary spectroscopic tool to monitor molecular structure and dynamics at atomic resolution in a complex setting (see ref. 6 for a recent review). Indeed, ssNMR has already been used to study individual molecular components in the context of natural bilayers (7,8), bacterial cell walls (9), and cellular organelles (10).Here, we introduce a general approach to investigate structure and dynamics of an arbitrary molecular target and its potential molecular partners in a cellular setting. Our studies focuses on the Gram-negative bacterial cell that is characterized by a molecularly complex but architecturally unique envelope, consisting of two lipid bilayers, the inner and outer membrane (IM, OM), separated by the periplasm containing the peptidoglycan (PG) layer (Fig. 1A). The IM is a phospholipid bilayer and harbors α-helical proteins, whereas the OM is asymmetrical and consists of phospholipids, lipopolysaccharides (LPS), lipoproteins, and β-barrelfold integral membrane proteins. LPS forms the outermost layer of the OM and protects the cell against harmful compounds from the environment. PG is a large macromolecule that gives the cell its shape and rigidity.Using uniformly 13 C, 15 N-labeled cellular preparations of Escherichia coli, we characterized the structure and dynamics of a recombinant integral membrane protein (PagL) and other major endogenous molecular components of the cell envelope in...

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Section: Discussionmentioning

confidence: 99%

Cellular solid-state nuclear magnetic resonance spectroscopy

Renault¹,

Boxtel²,

Bos³

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

180

220

View full text Add to dashboard Cite

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“…8,9 Materials and biological systems such as membrane or microcrystalline proteins, [10][11][12][13] fibrils, 14 ligands bound to receptors, 15 polymers, [16][17][18] surface-bound species, [19][20][21][22][23] and other low concentrated samples were studied beneficially using DNP-NMR. 13,[24][25][26] Cryogenic temperatures are required to slow down nuclear and electron relaxation and to allow an efficient polarization transfer in solid-state DNP NMR experiments. In contrast to biological systems, 15,[27][28][29] most technically relevant materials do not show detrimental lines broadening at low temperatures.…”

Section: A Introductionmentioning

confidence: 99%

Dynamic nuclear polarization of spherical nanoparticles

Akbey

Altın

Linden

et al. 2013

Phys. Chem. Chem. Phys.

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Spherical silica nanoparticles of various particle sizes (B10 to 100 nm), produced by a modified Stoeber method employing amino acids as catalysts, are investigated using Dynamic Nuclear Polarization (DNP) enhanced Nuclear Magnetic Resonance (NMR) spectroscopy. This study includes ultra-sensitive detection of surface-bound amino acids and their supramolecular organization in trace amounts, exploiting the increase in NMR sensitivity of up to three orders of magnitude via DNP. Moreover, the nature of the silicon nuclei on the surface and the bulk silicon nuclei in the core (sub-surface) is characterized at atomic resolution. Thereby, we obtain unique insights into the surface chemistry of these nanoparticles, which might result in improving their rational design as required for promising applications, e.g. as catalysts or imaging contrast agents. The non-covalent binding of amino acids to surfaces was determined which shows that the amino acids not just function as catalysts but become incorporated into the nanoparticles during the formation process. As a result only three distinct Q-types of silica signals were observed from surface and core regions. We observed dramatic changes of DNP enhancements as a function of particle size, and very small particles (which suit in vivo applications better) were hyperpolarized with the best efficiency. Nearly one order of magnitude larger DNP enhancement was observed for nanoparticles with 13 nm size compared to particles with 100 nm size.We determined an approximate DNP penetration-depth (B4.2 or B5.7 nm) for the polarization transfer from electrons to the nuclei of the spherical nanoparticles. Faster DNP polarization buildup was observed for larger nanoparticles. Efficient hyperpolarization of such nanoparticles, as achieved in this work, can be utilized in applications such as magnetic resonance imaging (MRI). A IntroductionDNP increases the sensitivity of NMR experiments (signal-to-noise ratio, S/N), 1-3 enabling the study of low concentrated systems which were not in the scope of NMR until now. NMR signal enhancement is achieved by transferring large electron polarization to surrounding nuclei via DNP. In the last decade, there has been remarkable progress in solution and solid-state DNP-NMR as well as in imaging through the application of hyperpolarization methods. [4][5][6][7] As pointed out recently, this ''renaissance of DNP'' combined with high magnetic fields will further grow in the coming years, enabling demanding applications with better resolution. 8,9 Materials and biological systems such as membrane or microcrystalline proteins, 10-13 fibrils, 14 ligands bound to receptors, 15 polymers, [16][17][18] surface-bound species, 19-23 and other low concentrated samples were studied beneficially using DNP-NMR. 13,[24][25][26] Cryogenic temperatures are required to slow down nuclear and electron relaxation and to allow an efficient polarization transfer in solid-state DNP NMR experiments. In contrast to biological systems, 15,27-29 most technically relevant materials ...

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“…Although hyperpolarization techniques made numerous new NMR applications possible, 6,[9][10][11][12][13][14][15][16][17] there are still many problems to be solved in this field. In particular, it is of importance to further develop strategies for optimizing the hyperpolarization process and the transfer of polarization from the primarily polarized spins to the target nuclei of choice.…”

Section: Introductionmentioning

confidence: 99%

Exploiting level anti-crossings for efficient and selective transfer of hyperpolarization in coupled nuclear spin systems

Pravdivtsev

Yurkovskaya

Kaptein

et al. 2013

Phys. Chem. Chem. Phys.

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Spin hyperpolarization can be coherently transferred to other nuclei in field-cycling NMR experiments.At low magnetic fields spin polarization is redistributed in a strongly coupled network of spins.Polarization transfer is most efficient at fields where level anti-crossings (LACs) occur for the nuclear spin-states. A further condition is that field switching to the LAC positions is non-adiabatic in order to convert the starting population differences into spin coherences that cause time-dependent mixing of states. The power of this method has been demonstrated by studying transfer of photo-Chemically Induced Dynamic Nuclear Polarization (photo-CIDNP) in N-acetyl-tryptophan. We have investigated the magnetic field dependence and time dependence of coherent CIDNP transfer and directly assessed nuclear spin LACs by studying polarization transfer at specific field positions. The proposed approach based on LACs is not limited to CIDNP but is advantageous for enhancing NMR signals by spin order transfer from any type of hyper-polarized nuclei.

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Solid‐State NMR Spectroscopy on Cellular Preparations Enhanced by Dynamic Nuclear Polarization

Cited by 170 publications

References 24 publications

Cellular solid-state nuclear magnetic resonance spectroscopy

Cellular solid-state nuclear magnetic resonance spectroscopy

Dynamic nuclear polarization of spherical nanoparticles

Exploiting level anti-crossings for efficient and selective transfer of hyperpolarization in coupled nuclear spin systems

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