Dynamic Nuclear Polarization Magic-Angle Spinning Nuclear Magnetic Resonance Combined with Molecular Dynamics Simulations Permits Detection of Order and Disorder in Viral Assemblies #DNPNMR
Gupta, Rupal, Huilan Zhang, Manman Lu, Guangjin Hou, Marc Caporini, Melanie Rosay, Werner Maas, et al. “Dynamic Nuclear Polarization Magic-Angle Spinning Nuclear Magnetic Resonance Combined with Molecular Dynamics Simulations Permits Detection of Order and Disorder in Viral Assemblies.” The Journal of Physical Chemistry B 123, no. 24 (June 20, 2019): 5048–58.
We report dynamic nuclear polarization (DNP) enhanced magic angle spinning (MAS) NMR spectroscopy in viral capsids from HIV-1 and bacteriophage AP205. Viruses regulate their lifecycles and infectivity through modulation of their structures and dynamics. While static structures of capsids from several viruses are now accessible with near-atomic level resolution, atomic-level understanding of functionally important motions in assembled capsids is lacking. We observed up to 64-fold signal enhancements by DNP, which permitted in-depth analysis of these assemblies. For the HIV-1 CA assemblies, remarkably high spectral resolution in the 3D and 2D heteronuclear datasets permitted the assignments of a significant fraction of backbone and side chain resonances. Using an integrated DNP MAS NMR and molecular dynamics simulations approach, the conformational space sampled by the assembled CA at cryogenic temperatures was mapped. Qualitatively, remarkable agreement was observed for the experimental 13C/15N chemical shift distributions and those calculated from substructures along the MD trajectory. Residues that are mobile at physiological temperatures are frozen out in multiple conformers at cryogenic conditions, resulting in broad experimental and calculated chemical shift distributions. Overall, our results suggest that DNP MAS NMR measurements in combination with MD simulations facilitate a thorough understanding of the dynamic signatures of viral capsids.
Post-doctoral position in MAS-DNP for the study of bacterial cell wall at CEA / Univ. Grenoble Alpes (France)
A post-doctoral position is available immediately for a period of 18 months to work with Sabine Hediger and Gaël De Paëpe on developing MAS-DNP for the study of bacterial cell wall. More information about our group at CEA Grenoble / Univ. Grenoble Alpes and a list of recent publications can be found here: www.dnpgrenoble.net.
Context of the project:
For over 50 years, peptidoglycan has played a pivotal role in the development of antibacterial chemotherapy. In the hunt for new drugs, the biosynthetic pathways of this ubiquitous cell wall polymer have been deciphered and essential peptidoglycan-synthesizing enzymes identified as antibacterial targets with high potential. Focusing on the L,D-transpeptidation, a key cell-wall synthesis and maturation reaction, we will first study purified enzymes in interaction with peptidoglycan fragments and then move on to the study of the complete cell-wall synthesis machinery in bacterial cells during the cell maturation.
In this context, innovative spectroscopic approaches including MAS-DNP will be conducted to provide new tools for the investigation of protein interaction with the bacterial cell wall. This project aims at developing the available MAS-DNP technique in order to provide best spectral sensitivity and resolution for the investigation of the bacterial cell wall itself in extracted and entire cells, as well as possible interactions with proteins involved in L,D-transpeptidation. These developments will make use of state-of-the-art DNP methods and polarizing agents developed or under development in the lab, which hosts two 400 MHz MAS-DNP spectrometers.
Requirements and application:
Applicants are expected to have a doctoral degree in liquid-state and/or solid-state biomolecular NMR spectroscopy. Knowledge about MAS-DNP will be considered as a plus. The successful candidate will be recruited for 18 months and will benefit from an ANR postdoctoral fellowship. Deadline for application is end of August. Interested candidates are welcomed to send an email to: firstname.lastname@example.org
Grenoble is one of the major cities in Europe for research with a large international scientific community. In addition, Grenoble has a large international student population, is a very pleasant city to live in, and is known as the “Capital of the Alps” with easy access to great skiing and hiking. It’s also only 2 hours’ drive to the Mediterranean Sea, Italy, or Switzerland. Grenoble, Lyon, and Geneva airports are nearby and permit straightforward international travel.
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Dynamic Nuclear Polarization / solid-state NMR of membranes. Thermal effects and sample geometry #DNPNMR
Salnikov, Evgeniy Sergeevich, Fabien Aussenac, Sebastian Abel, Armin Purea, Paul Tordo, Olivier Ouari, and Burkhard Bechinger. “Dynamic Nuclear Polarization / Solid-State NMR of Membranes. Thermal Effects and Sample Geometry.” Solid State Nuclear Magnetic Resonance 100 (August 2019): 70–76.
Whereas specially designed dinitroxide biradicals, reconstitution protocols, oriented sample geometries and NMR probes have helped to much increase the DNP enhancement factors of membrane samples they still lag considerably behind those obtained from glasses made of protein solutions. Here we show that not only the MAS rotor material but also the distribution of the membrane samples within the NMR rotor have a pronounced effect on the DNP efficiency. These observations are rationalized with the cooling efficiency and the internal properties of the sample, monitored by their T1 relaxation, microwave on versus off signal intensities and DNP enhancement. The data are suggestive that for membranes the speed of cooling has a pronounced effect on membrane phase transitions and concomitantly the distribution of biradicals within the sample.
Purea, Armin, Christian Reiter, Alexandros I. Dimitriadis, Emile de Rijk, Fabien Aussenac, Ivan Sergeyev, Melanie Rosay, and Frank Engelke. “Improved Waveguide Coupling for 1.3 Mm MAS DNP Probes at 263 GHz.” Journal of Magnetic Resonance 302 (May 2019): 43–49.
We consider the geometry of a radially irradiated microwave beam in MAS DNP NMR probes and its impact on DNP enhancement. Two related characteristic features are found to be relevant: (i) the focus of the microwave beam on the DNP MAS sample and (ii) the microwave magnetic field magnitude in the sample. We present a waveguide coupler setup that enables us to significantly improve beam focus and field magnitude in 1.3 mm MAS DNP probes at a microwave frequency of 263 GHz, which results in an increase of the DNP enhancement by a factor of 2 compared to previous standard hardware setups. We discuss the implications of improved coupling and its potential to enable cutting-edge applications, such as pulsed high-field DNP and the use of low-power solid-state microwave sources.
Lund, Alicia, Asif Equbal, and Songi Han. “Tuning Nuclear Depolarization under MAS by Electron T1e.” Physical Chemistry Chemical Physics, 2018, 19.
Cross-Effect (CE) Dynamic Nuclear Polarization (DNP) mechanism under Magic Angle Spinning (MAS) induces depletion or “depolarization” of the NMR signal, in the absence of microwave irradiation. In this study, the role of T1e on nuclear depolarization under MAS was tested experimentally by systematically varying the local and global electron spin concentration using mono-, bi- and tri-radicals. These spin systems show different depolarization effects that systematically tracked with their different T1e rates, consistent with theoretical predictions. In order to test whether the effect of T1e is directly or indirectly convoluted with other spin parameters, the tri-radical system was doped with different concentrations of GdCl3, only tuning the T1e rates, while keeping other parameters unchanged. Gratifyingly, the changes in the depolarization factor tracked the changes in the T1e rates. The experimental results are corroborated by quantum mechanics based numerical simulations which recapitulated the critical role of T1e. Simulations showed that the relative orientation of the two g-tensors and e-e dipolar interaction tensors of the CE fulfilling spin pair also plays a major role in determining the extent of depolarization, besides the enhancement. This is expected as orientations influence the efficiency of the various level anti-crossings or the “rotor events” under MAS. However, experimental evaluation of the empirical spectral diffusion parameter at static condition showed that the local vs. global e-e dipolar interaction network is not a significant variable in the commonly used nitroxide radical system studied here, leaving T1e rates as the major modulator of depolarization.
Dynamic Nuclear Polarization-Enhanced Biomolecular NMR Spectroscopy at High Magnetic Field with Fast Magic-Angle Spinning #DNPNMR
Jaudzems, Kristaps, Andrea Bertarello, Sachin R. Chaudhari, Andrea Pica, Diane Cala-De Paepe, Emeline Barbet-Massin, Andrew J. Pell, et al. “Dynamic Nuclear Polarization-Enhanced Biomolecular NMR Spectroscopy at High Magnetic Field with Fast Magic-Angle Spinning.” Angewandte Chemie 0 (2018).
Dynamic nuclear polarization (DNP) is a powerful way to overcome the sensitivity limitation of magic?angle?spinning (MAS) NMR experiments. However, the resolution of the DNP?NMR spectra of proteins is compromised by severe line broadening associated with the necessity to perform experiments at cryogenic temperatures and in the presence of paramagnetic radicals. High?quality DNP?enhanced NMR spectra of the Acinetobacter phage 205 (AP205) nucleocapsid can be obtained by combining high magnetic field (800?MHz) and fast MAS (40?kHz). These conditions yield enhanced resolution and long coherence lifetimes allowing the acquisition of resolved 2D correlation spectra and of previously unfeasible scalar?based experiments. This enables the assignment of aromatic resonances of the AP205 coat protein and its packaged RNA, as well as the detection of long?range contacts, which are not observed at room temperature, opening new possibilities for structure determination.
Instrumentation for cryogenic magic angle spinning dynamic nuclear polarization using 90L of liquid nitrogen per day #DNPNMR
Albert, B.J., et al., Instrumentation for cryogenic magic angle spinning dynamic nuclear polarization using 90L of liquid nitrogen per day. J. Magn. Reson., 2017. 283(Supplement C): p. 71-78.
Cryogenic sample temperatures can enhance NMR sensitivity by extending spin relaxation times to improve dynamic nuclear polarization (DNP) and by increasing Boltzmann spin polarization. We have developed an efficient heat exchanger with a liquid nitrogen consumption rate of only 90L per day to perform magic-angle spinning (MAS) DNP experiments below 85K. In this heat exchanger implementation, cold exhaust gas from the NMR probe is returned to the outer portion of a counterflow coil within an intermediate cooling stage to improve cooling efficiency of the spinning and variable temperature gases. The heat exchange within the counterflow coil is calculated with computational fluid dynamics to optimize the heat transfer. Experimental results using the novel counterflow heat exchanger demonstrate MAS DNP signal enhancements of 328±3 at 81±2K, and 276±4 at 105±2K.
Mentink-Vigier, F., S. Vega, and G. De Paepe, Fast and accurate MAS-DNP simulations of large spin ensembles. Phys. Chem. Chem. Phys., 2017. 19(5): p. 3506-3522.
A deeper understanding of parameters affecting Magic Angle Spinning Dynamic Nuclear Polarization (MAS-DNP), an emerging nuclear magnetic resonance hyperpolarization method, is crucial for the development of new polarizing agents and the successful implementation of the technique at higher magnetic fields (>10 T). Such progress is currently impeded by computational limitation which prevents the simulation of large spin ensembles (electron as well as nuclear spins) and to accurately describe the interplay between all the multiple key parameters at play. In this work, we present an alternative approach to existing cross-effect and solid-effect MAS-DNP codes that yields fast and accurate simulations. More specifically we describe the model, the associated Liouville-based formalism (Bloch-type derivation and/or Landau-Zener approximations) and the linear time algorithm that allows computing MAS-DNP mechanisms with unprecedented time savings. As a result, one can easily scan through multiple parameters and disentangle their mutual influences. In addition, the simulation code is able to handle multiple electrons and protons, which allows probing the effect of (hyper)polarizing agents concentration, as well as fully revealing the interplay between the polarizing agent structure and the hyperfine couplings, nuclear dipolar couplings, nuclear relaxation times, both in terms of depolarization effect, but also of polarization gain and buildup times.
Ni, Q.Z., et al., Peptide and Protein Dynamics and Low-Temperature/DNP Magic Angle Spinning NMR. J Phys Chem B, 2017.
In DNP MAS NMR experiments at ~80-110 K, the structurally important -13CH3 and -15NH3+ signals in MAS spectra of biological samples disappear due to the interference of the molecular motions with the 1H decoupling. Here we investigate the effect of these dynamic processes on the NMR lineshapes and signal intensities in several typical systems: (1) microcrystalline APG, (2) membrane protein bR, (3) amyloid fibrils PI3-SH3, (4) monomeric alanine-CD3 and (5) the pro-tonated and deuterated dipeptide N-Ac-VL over 78-300 K. In APG, the 3-site hopping of the Ala-Cbeta peak disappears com-pletely at 112 K, concomitant with the attenuation of CP signals from other 13C\’s and 15N\’s. Similarly, the 15N signal from Ala-NH3+ disappears ~173 K, concurrent with the attenuation in CP experiments of other 15N\’s as well as 13C\’s. In bR and PI3-SH3, the methyl groups are attenuated at ~95 K while all other 13C\’s remain unaffected. However, both systems exhibit substantial losses of intensity at ~243 K. Finally, with spectra of Ala and N-Ac-VL we show that it is possible to extract site specific dynamic data from the temperature dependence of the intensity losses. Furthermore, 2H labeling can assist with re-covering the spectral intensity. Thus, our study provides insight into the dynamic behavior of biological systems over a wide range of temperatures, and serves as a guide to optimizing the sensitivity and resolution of structural data in low temperature DNP MAS NMR spectra.
Joint Postdoctoral position available in biomolecular NMR and DNP in Grenoble (France)
In vitro and in vivo NMR investigation of the L,D-transpeptidase:peptidoglycan complex and of the mycobacterial cell-wall maturation
For over 50 years, peptidoglycan has played a pivotal role in the development of antibacterial chemotherapy, and essential peptidoglycan-synthesizing enzymes have been identified as antibacterial targets with high potential and characterized in vitro. Nevertheless the efforts to develop drugs acting on these rationally chosen targets have largely proven disappointing due to the limited number of biophysical tools capable to produce static and dynamic structural views of the entire peptidoglycan polymer along the bacterial cell-life cycle and of its evolution under the selective pressure of antibiotics. Focusing on one important cell-wall synthesis and maturation reaction, the L,D-transpeptidation, the present project aims at combining information obtained on samples of different levels of complexity, ranging from purified enzymes in interaction with peptidoglycan fragments to the complete cell-wall synthesis machinery in bacterial cells. In this context, innovative spectroscopic approaches including high-field solution NMR, solid-state NMR as well as MAS-DNP will be conducted to provide a new view on the role of L,D-transpeptidases (Ldts) in the cross-linking of peptidoglycan peptide stems along the cell maturation.
To succeed in this integrative approach a joint postdoctoral position is proposed between two NMR research groups in Grenoble that already collaborated efficiently in the past. Structural and dynamical studies of peptidoglycan:L,D-transpeptidase complexes by liquid- and solid-state NMR spectroscopy will be mainly hosted in the Biomolecular NMR Spectroscopy group at Institut de Biologie Structurale (IBS, http://www.ibs.fr/) in the team directed by Dr Jean-Pierre Simorre. This group has a direct access to the state of the art NMR facility at IBS containing six high-field spectrometers (950 MHz, 850 MHz, 700 MHz, 3×600 MHz) equipped with latest solid-state NMR and cryogenic liquid-state probes. Furthermore, an innovative approach based on MAS-DNP will be developed, in particular for in-vivo studies. This part of the work will take place in the DNP group of the Institute for Nanosciences and Cryogenics at CEA Grenoble under the supervision of Sabine Hediger. This team, directed by Gaël De Paëpe, hosts a 400 MHz MAS-DNP spectrometer and is very active on instrumentation and methods developments in standard and ultra-low temperature MAS-DNP. The synergy between both groups is reinforced by the geographical proximity.
Applicants are expected to have a doctoral experience in liquid-state and/or solid-state biomolecular NMR spectroscopy. Knowledge about MAS-DNP will be considered as a plus. The successful candidate will be recruited for 18 months and will benefit from an ANR postdoctoral fellowship. Interested candidates should send their application with a curriculum vitae, a letter of motivation, and 2 reference letters by May 31st, 2017 via email to Jean-Pierre Simorre (email@example.com) and Sabine Hediger (firstname.lastname@example.org).
17 rue des Martyrs
38054 Grenoble Cedex 9
Tel.: +33 4 38 78 65 79
Fax : +33 4 38 78 50 90
Email : email@example.com
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