Category Archives: Structural Biology

Heteronuclear cross-relaxation effect modulated by the dynamics of N-functional groups in the solid state under 15N DP-MAS DNP #DNPNMR

Park, Heeyong, Boran Uluca-Yazgi, Saskia Heumann, Robert Schlögl, Josef Granwehr, Henrike Heise, and P. Philipp M. Schleker. “Heteronuclear Cross-Relaxation Effect Modulated by the Dynamics of N-Functional Groups in the Solid State under 15N DP-MAS DNP.” Journal of Magnetic Resonance 312 (March 2020): 106688.

https://doi.org/10.1016/j.jmr.2020.106688

In a typical magic-angle spinning (MAS) dynamic nuclear polarization (DNP) nuclear magnetic resonance (NMR) experiment, several mechanisms are simultaneously involved when transferring much larger polarization of electron spins to NMR active nuclei of interest. Recently, specific cross-relaxation enhancement by active motions under DNP (SCREAM-DNP) [Daube et al. JACS 2016] has been reported as one of these mechanisms. Thereby 13C enhancement with inverted sign was observed in a direct polarization (DP) MAS DNP experiment, caused by reorientation dynamics of methyl that was not frozen out at 100 K. Here, we report on the spontaneous polarization transfer from hyperpolarized 1H to both primary amine and ammonium nitrogens, resulting in an additional positive signal enhancement in the 15N NMR spectra during 15N DP-MAS DNP. The cross-relaxation induced signal enhancement (CRE) for 15N is of opposite sign compared to that observed for 13C due to the negative sign of the gyromagnetic ratio of 15N. The influence on CRE efficiency caused by variation of the radical solution composition and by temperature was also investigated.

DNP and Cellular Solid-state NMR #DNPNMR

Paioni, Alessandra Lucini, Marie A M Renault, and Marc Baldus. “DNP and Cellular Solid-State NMR,” 7:12, 2018.

https://onlinelibrary.wiley.com/doi/abs/10.1002/9780470034590.emrstm1561.

Solid‐state nuclear magnetic resonance (ssNMR) can provide structural information at the most detailed level and, at the same time, is applicable in highly heterogeneous and complex molecular environments, largely irrespective of solubility or crystallinity. Revolutionary developments in the field of dynamic nuclear polarization (DNP) have greatly enhanced ssNMR sensitivity. In this article, we discuss ssNMR concepts and applications that make use of these advancements and enable the study of complex biomolecular and even cellular systems at unprecedented structural resolution and molecular detail.

Photocycle-dependent conformational changes in the proteorhodopsin cross-protomer Asp–His–Trp triad revealed by DNP-enhanced MAS-NMR #DNPNMR

Maciejko, Jakob, Jagdeep Kaur, Johanna Becker-Baldus, and Clemens Glaubitz. “Photocycle-Dependent Conformational Changes in the Proteorhodopsin Cross-Protomer Asp–His–Trp Triad Revealed by DNP-Enhanced MAS-NMR.” Proceedings of the National Academy of Sciences 116, no. 17 (April 23, 2019): 8342–49.

https://doi.org/10.1073/pnas.1817665116

Proteorhodopsin (PR) is a highly abundant, pentameric, light-driven proton pump. Proton transfer is linked to a canonical photocycle typical for microbial ion pumps. Although the PR monomer is able to undergo a full photocycle, the question arises whether the pentameric complex formed in the membrane via specific cross-protomer interactions plays a role in its functional mechanism. Here, we use dynamic nuclear polarization (DNP)-enhanced solid-state magic-angle spinning (MAS) NMR in combination with light-induced cryotrapping of photointermediates to address this topic. The highly conserved residue H75 is located at the protomer interface. We show that it switches from the (τ)- to the (π)-tautomer and changes its ring orientation in the M state. It couples to W34 across the oligomerization interface based on specific His/Trp ring orientations while stabilizing the pKa of the primary proton acceptor D97 within the same protomer. We further show that specific W34 mutations have a drastic effect on D97 and proton transfer mediated through H75. The residue H75 defines a cross-protomer Asp–His–Trp triad, which potentially serves as a pH-dependent regulator for proton transfer. Our data represent light-dependent, functionally relevant cross talk between protomers of a microbial rhodopsin homo-oligomer.

DNP NMR of biomolecular assemblies #DNPNMR

Jaudzems, Kristaps, Tatyana Polenova, Guido Pintacuda, Hartmut Oschkinat, and Anne Lesage. “DNP NMR of Biomolecular Assemblies.” Journal of Structural Biology 206, no. 1 (April 2019): 90–98.

https://doi.org/10.1016/j.jsb.2018.09.011

Dynamic Nuclear Polarization (DNP) is an effective approach to alleviate the inherently low sensitivity of solid-state NMR (ssNMR) under magic angle spinning (MAS) towards large-sized multi-domain complexes and assemblies. DNP relies on a polarization transfer at cryogenic temperatures from unpaired electrons to adjacent nuclei upon continuous microwave irradiation. This is usually made possible via the addition in the sample of a polarizing agent. The first pioneering experiments on biomolecular assemblies were reported in the early 2000s on bacteriophages and membrane proteins. Since then, DNP has experienced tremendous advances, with the development of extremely efficient polarizing agents or with the introduction of new microwaves sources, suitable for NMR experiments at very high magnetic fields (currently up to 900 MHz). After a brief introduction, several experimental aspects of DNP enhanced NMR spectroscopy applied to biomolecular assemblies are discussed. Recent demonstration experiments of the method on viral capsids, the type III and IV bacterial secretion systems, ribosome and membrane proteins are then described.

DNP NMR of biomolecular assemblies #DNPNMR

Jaudzems, Kristaps, Tatyana Polenova, Guido Pintacuda, Hartmut Oschkinat, and Anne Lesage. “DNP NMR of Biomolecular Assemblies.” Journal of Structural Biology 206, no. 1 (April 2019): 90–98.

https://doi.org/10.1016/j.jsb.2018.09.011.

Dynamic Nuclear Polarization (DNP) is an effective approach to alleviate the inherently low sensitivity of solid-state NMR (ssNMR) under magic angle spinning (MAS) towards large-sized multi-domain complexes and assemblies. DNP relies on a polarization transfer at cryogenic temperatures from unpaired electrons to adjacent nuclei upon continuous microwave irradiation. This is usually made possible via the addition in the sample of a polarizing agent. The first pioneering experiments on biomolecular assemblies were reported in the early 2000s on bacteriophages and membrane proteins. Since then, DNP has experienced tremendous advances, with the development of extremely efficient polarizing agents or with the introduction of new microwaves sources, suitable for NMR experiments at very high magnetic fields (currently up to 900 MHz). After a brief introduction, several experimental aspects of DNP enhanced NMR spectroscopy applied to biomolecular assemblies are discussed. Recent demonstration experiments of the method on viral capsids, the type III and IV bacterial secretion systems, ribosome and membrane proteins are then described.

Structure determination of supra-molecular assemblies by solid-state NMR: Practical considerations #DNPNMR

Demers, Jean-Philippe, Pascal Fricke, Chaowei Shi, Veniamin Chevelkov, and Adam Lange. “Structure Determination of Supra-Molecular Assemblies by Solid-State NMR: Practical Considerations.” Progress in Nuclear Magnetic Resonance Spectroscopy 109 (December 2018): 51–78.

https://doi.org/10.1016/j.pnmrs.2018.06.002

In the cellular environment, biomolecules assemble in large complexes which can act as molecular machines. Determining the structure of intact assemblies can reveal conformations and inter-molecular interactions that are only present in the context of the full assembly. Solid-state NMR (ssNMR) spectroscopy is a technique suitable for the study of samples with high molecular weight that allows the atomic structure determination of such large protein assemblies under nearly physiological conditions.

This review provides a practical guide for the first steps of studying biological supramolecular assemblies using ssNMR. The production of isotope-labeled samples is achievable via several means, which include recombinant expression, cell-free protein synthesis, extraction of assemblies directly from cells, or even the study of assemblies in whole cells in situ. Specialized isotope labeling schemes greatly facilitate the assignment of chemical shifts and the collection of structural data. Advanced strategies such as mixed, diluted, or segmental subunit labeling offer the possibility to study inter-molecular interfaces.

Detailed and practical considerations are presented with respect to first setting up magicangle spinning (MAS) ssNMR experiments, including the selection of the ssNMR rotor, different methods to best transfer the sample and prepare the rotor, as well as common and robust procedures for the calibration of the instrument. Diagnostic spectra to evaluate the resolution and sensitivity of the sample are presented. Possible improvements that can reduce sample heterogeneity and improve the quality of ssNMR spectra are reviewed.

High-sensitivity protein solid-state NMR spectroscopy #DNPNMR

Mandala, Venkata S, and Mei Hong. “High-Sensitivity Protein Solid-State NMR Spectroscopy.” Current Opinion in Structural Biology 58 (October 2019): 183–90.

https://doi.org/10.1016/j.sbi.2019.03.027

The sensitivity of solid-state nuclear magnetic resonance (SSNMR) spectroscopy for structural biology is significantly increased by 1H detection under fast magic-angle spinning (MAS) and by dynamic nuclear polarization (DNP) from electron spins to nuclear spins. The former allows studies of the structure and dynamics of small quantities of proteins under physiological conditions, while the latter permits studies of large biomolecular complexes in lipid membranes and cells, protein intermediates, and protein conformational distributions. We highlight recent applications of these two emerging SSNMR technologies and point out areas for future development.

DNP-Enhanced MAS NMR: A Tool to Snapshot Conformational Ensembles of α-Synuclein in Different States #DNPNMR

Uluca, Boran, Thibault Viennet, Dušan Petrović, Hamed Shaykhalishahi, Franziska Weirich, Ayşenur Gönülalan, Birgit Strodel, Manuel Etzkorn, Wolfgang Hoyer, and Henrike Heise. “DNP-Enhanced MAS NMR: A Tool to Snapshot Conformational Ensembles of α-Synuclein in Different States.” Biophysical Journal 114, no. 7 (April 2018): 1614–23.

https://doi.org/10.1016/j.bpj.2018.02.011.

Intrinsically disordered proteins dynamically sample a wide conformational space and therefore do not adopt a stable and defined three-dimensional conformation. The structural heterogeneity is related to their proper functioning in physiological processes. Knowledge of the conformational ensemble is crucial for a complete comprehension of this kind of proteins. We here present an approach that utilizes dynamic nuclear polarization-enhanced solid-state NMR spectroscopy of sparsely isotope-labeled proteins in frozen solution to take snapshots of the complete structural ensembles by exploiting the inhomogeneously broadened line-shapes. We investigated the intrinsically disordered protein a-synuclein (a-syn), which plays a key role in the etiology of Parkinson’s disease, in three different physiologically relevant states. For the free monomer in frozen solution we could see that the so-called ‘‘random coil conformation’’ consists of a-helical and b-sheet-like conformations, and that secondary chemical shifts of neighboring amino acids tend to be correlated, indicative of frequent formation of secondary structure elements. Based on these results, we could estimate the number of disordered regions in fibrillar a-syn as well as in a-syn bound to membranes in different protein-to-lipid ratios. Our approach thus provides quantitative information on the propensity to sample transient secondary structures in different functional states. Molecular dynamics simulations rationalize the results.

Postdoctoral position: DNP-enhanced biomolecular solid-state NMR-spectroscopy for the study of protein (mis)folding #DNPNMR

A postdoctoral position is available in the group of Henrike Heise at Forschungszentrum Juelich/Heinrich Heine University Duesseldorf

The successful candidate will study disordered and aggregation-prone proteins in all stages of the misfolding pathway. Further, interaction with potential drug candidates will be elucidated. State-of-the art solid-state NMR-spectroscopy, involvng polarization enhancement by DNP and fast magic angle spinning will be applied to elucidate those processes at atomic resolution. A selection of relevant publication is given below.

Our laboratory at the Forschungszentrum Jülich is equipped with state of the art solid-state NMR spectrometers operating at 800 and 600 MHz, DNP equipment is available for 600 and 800 MHz spectrometers. An ultrafast MAS probe head for spinning speeds up to > 100 kHz will be available soon, the installation of an ultrahigh field spectrometer operating at 1.2 GHz is scheduled for 2020.

The Forschungszentrum Jülich offers a vibrant research environment with close collaborations with the University of Düsseldorf, the RWTH Aachen University and the Max-Planck Institute Mülheim. Our laboratory is part of the Jülich center of structural biology JuStruct which bundles expertise with infrastructure in the field of atomic resolution of structural biology (NMR in solution and the solid state, X-ray crystallography,cryo-EM, molecular modelling and neutron scattering).

The initial appointment is for a year, with a possibility of renewal.The initial appointment is for a year, with a possibility of renewal.

Applications will be reviewed on a rolling basis, and candidates will be considered until the position is filled.

The ideal candidate should have a strong background in biomolecular NMR-spectroscopy. Experience with hyperpolarization and/ or Protein expression and purfication is a plus. Applicants must submit a cover letter summarizing research experience and specifying the interests in this position; a curriculum vitae (including a publication list); a statement of research interests; and two letters of reference to henrike.heise@hhu.de

Information about our group:

http://www.fknmr.hhu.de/en/

Selection of relevant publications:

  • L. Siemons, B. Uluca-Yazgi, R. B. Pritchard, S. McCarthy, H. Heise, D. F. Hansen, Determining isoleucine side-chain rotamer-sampling in proteins from 13C chemical shift, Chem.Commun. 2019, in press. doi:10.1039/C9CC06496F.
  • A. König, D. Schölzel, B. Uluca, T. Viennet, Ü. Akbey, H. Heise, Hyperpolarized MAS NMR of unfolded and misfolded proteins, Solid State NMR, 2019, 98, 1-11.
  • B. Uluca, T. Viennet, D. Petrović, H. Shaykhalishahi, F. Weirich, A. Gönülalan, B. Strodel, M. Etzkorn, W. Hoyer, and H. Heise. DNP-Enhanced solid-state NMR at Cryogenic Temperatures: a Tool to Snapshot Conformational Ensembles of α-Synuclein in Different States. Biophys. J. 2018, 114, 1614-1623.
  • L. Gremer, D. Schölzel, C. Schenk, E. Reinartz, J. Labahn, R. Ravelli, M. Tusche, C. Lopez-Iglesias, W. Hoyer, H. Heise, D. Willbold, G. Schröder, Fibril structure of amyloid-ß(1-42) by cryo-electron microscopy, Science, 2017, 358, 116-119.
  • T. Viennet, A. Viegas, A. Kuepper, S. Arens, V. Gelev, O. Petrov, T. N. Grossmann, H. Heise, M. Etzkorn, Selective Protein Hyperpolarization in Cell Lysates Using Targeted Dynamic Nuclear Polarization. Angew. Chem. Int. Ed. Engl. 2016, 55, 10746-10750.

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Forschungszentrum Juelich GmbH

52425 Juelich

Sitz der Gesellschaft: Juelich

Eingetragen im Handelsregister des Amtsgerichts Dueren Nr. HR B 3498

Vorsitzender des Aufsichtsrats: MinDir Volker Rieke

Geschaeftsfuehrung: Prof. Dr.-Ing. Wolfgang Marquardt (Vorsitzender),

Karsten Beneke (stellv. Vorsitzender), Prof. Dr.-Ing. Harald Bolt,

Prof. Dr. Sebastian M. Schmidt

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Dynamic Nuclear Polarization Nuclear Magnetic Resonance in Human Cells Using Fluorescent Polarizing Agents #DNPNMR

Albert, Brice J., Chukun Gao, Erika L. Sesti, Edward P. Saliba, Nicholas Alaniva, Faith J. Scott, Snorri Th. Sigurdsson, and Alexander B. Barnes. “Dynamic Nuclear Polarization Nuclear Magnetic Resonance in Human Cells Using Fluorescent Polarizing Agents.” Biochemistry 57, no. 31 (August 7, 2018): 4741–46.

https://doi.org/10.1021/acs.biochem.8b00257

Solid state nuclear magnetic resonance (NMR) enables atomic-resolution characterization of the molecular structure and dynamics within complex heterogeneous samples, but it is typically insensitive. Dynamic nuclear polarization (DNP) increases the NMR signal intensity by orders of magnitude and can be performed in combination with magic angle spinning (MAS) for sensitive, high-resolution spectroscopy. Here we report MAS DNP experiments, for the first time, within intact human cells with >40-fold DNP enhancement and a sample temperature of <6 K. In addition to cryogenic MAS results at <6 K, we also show in-cell DNP enhancements of 57-fold at 90 K. In-cell DNP is demonstrated using biradicals and sterically shielded monoradicals as polarizing agents. A novel trimodal polarizing agent is introduced for DNP, which contains a nitroxide biradical, a targeting peptide for cell penetration, and a fluorophore for subcellular localization with confocal microscopy. The fluorescent polarizing agent provides in-cell DNP enhancements of 63-fold at a concentration of 2.7 mM. These experiments pave the way for structural characterization of biomolecules in an endogenous cellular context.

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