Monday, August 31, 2015

Efficient Dynamic Nuclear Polarization at 800 MHz/527 GHz with Trityl-Nitroxide Biradicals


Mathies, G., et al., Efficient Dynamic Nuclear Polarization at 800 MHz/527 GHz with Trityl-Nitroxide Biradicals. Angew Chem Int Ed Engl, 2015: p. n/a-n/a.


Cross-effect (CE) dynamic nuclear polarization (DNP) is a rapidly developing technique that enhances the signal intensities in magic-angle spinning (MAS) NMR spectra. We report CE DNP experiments at 211, 600, and 800 MHz using a new series of biradical polarizing agents referred to as TEMTriPols, in which a nitroxide (TEMPO) and a trityl radical are chemically tethered. The TEMTriPol molecule with the optimal performance yields a record 1 H NMR signal enhancement of 65 at 800 MHz at a concentration of 10 mM in a glycerol/water solvent matrix. The CE DNP enhancement for the TEMTriPol biradicals does not decrease as the magnetic field is increased in the manner usually observed for bis-nitroxides. Instead, the relatively strong exchange interaction between the trityl and nitroxide moieties determines the magnetic field at which the optimum enhancement is observed.

Sunday, August 30, 2015

Meet Bridge12 at the 5th International DNP Symposium

Meet Thorsten Maly from Bridge12 Technologies, Inc. at the 5th International DNP Symposium in Egmond aan Zee (The Netherlands). The symposium will be held starting Monday August 31st and is ending Friday September 4th, 2015.

Dr. Maly will give a talk in the Instrumentation Session on Tuesday (8:30 - 12:30) giving an overview of our current research towards an Integrated Gyrotron System for DNP-NMR Spectroscopy.

Feel free to pull Dr. Maly aside if you would like to discuss your DNP-NMR spectroscopy applications or contact him at tmaly@bridge12.com.

Wednesday, August 26, 2015

Endogenous Stable Radicals for Characterization of Thermally Carbonized Porous Silicon by Solid-State Dynamic Nuclear Polarization 13C NMR


Riikonen, J., et al., Endogenous Stable Radicals for Characterization of Thermally Carbonized Porous Silicon by Solid-State Dynamic Nuclear Polarization13C NMR. The Journal of Physical Chemistry C, 2015. 119(33): p. 19272-19278.


As with all nanomaterials, characterization of the surface chemistry of mesoporous silicon (PSi) is crucial for the development in its diverse applications. Nuclear magnetic resonance (NMR) is one of the most powerful methods to study the chemistry of nanomaterials, but it is currently underutilized with PSi due to low signal-to-noise ratios achieved with this material which lead to very long measurement times. Here we show that endogenous radicals exist in thermally carbonized PSi and demonstrate the feasibility of solid-state dynamic nuclear polarization (DNP) NMR without addition of organic radicals. Use of DNP NMR is demonstrated to highly improve the signal-to-noise ratio while significantly reducing the measurement times. This technique opens new possibilities for the use of more advanced NMR techniques allowing the detailed characterization of complex materials such as PSi. Furthermore, the chemical structure of thermally carbonized PSi is studied by complementary techniques, X-ray photoelectron spectroscopy, Fourier transform infrared spectroscopy, and Raman spectroscopy.

Monday, August 24, 2015

Cysteine-Specific Labeling of Proteins with a Nitroxide Biradical for Dynamic Nuclear Polarization NMR


Voinov, M.A., et al., Cysteine-Specific Labeling of Proteins with a Nitroxide Biradical for Dynamic Nuclear Polarization NMR. J Phys Chem B, 2015. 119(32): p. 10180-90.


Dynamic nuclear polarization (DNP) enhances the signal in solid-state NMR of proteins by transferring polarization from electronic spins to the nuclear spins of interest. Typically, both the protein and an exogenous source of electronic spins, such as a biradical, are either codissolved or suspended and then frozen in a glycerol/water glassy matrix to achieve a homogeneous distribution. While the use of such a matrix protects the protein upon freezing, it also reduces the available sample volume (by ca. a factor of 4 in our experiments) and causes proportional NMR signal loss. Here we demonstrate an alternative approach that does not rely on dispersing the DNP agent in a glassy matrix. We synthesize a new biradical, ToSMTSL, which is based on the known DNP agent TOTAPOL, but also contains a thiol-specific methanethiosulfonate group to allow for incorporating this biradical into a protein in a site-directed manner. ToSMTSL was characterized by EPR and tested for DNP of a heptahelical transmembrane protein, Anabaena sensory rhodopsin (ASR), by covalent modification of solvent-exposed cysteine residues in two (15)N-labeled ASR mutants. DNP enhancements were measured at 400 MHz/263 GHz NMR/EPR frequencies for a series of samples prepared in deuterated and protonated buffers and with varied biradical/protein ratios. While the maximum DNP enhancement of 15 obtained in these samples is comparable to that observed for an ASR sample cosuspended with approximately 17 mM TOTAPOL in a glycerol-d8/D2O/H2O matrix, the achievable sensitivity would be 4-fold greater due to the gain in the filling factor. We anticipate that the DNP enhancements could be further improved by optimizing the biradical structure. The use of covalently attached biradicals would broaden the applicability of DNP NMR to structural studies of proteins.

Friday, August 21, 2015

Enlightening the photoactive site of channelrhodopsin-2 by DNP-enhanced solid-state NMR spectroscopy

Becker-Baldus, J., et al., Enlightening the photoactive site of channelrhodopsin-2 by DNP-enhanced solid-state NMR spectroscopy. Proc. Nat. Aca. Sci. USA, 2015.


Channelrhodopsin-2 from Chlamydomonas reinhardtii is a light-gated ion channel. Over recent years, this ion channel has attracted considerable interest because of its unparalleled role in optogenetic applications. However, despite considerable efforts, an understanding of how molecular events during the photocycle, including the retinal trans-cis isomerization and the deprotonation/reprotonation of the Schiff base, are coupled to the channel-opening mechanism remains elusive. To elucidate this question, changes of conformation and configuration of several photocycle and conducting/nonconducting states need to be determined at atomic resolution. Here, we show that such data can be obtained by solid-state NMR enhanced by dynamic nuclear polarization applied to 15N-labeled channelrhodopsin-2 carrying 14,15-13C2 retinal reconstituted into lipid bilayers. In its dark state, a pure all-trans retinal conformation with a stretched C14-C15 bond and a significant out-of-plane twist of the H-C14-C15-H dihedral angle could be observed. Using a combination of illumination, freezing, and thermal relaxation procedures, a number of intermediate states was generated and analyzed by DNP-enhanced solid-state NMR. Three distinct intermediates could be analyzed with high structural resolution: the early P1500 K-like state, the slowly decaying late intermediate P4480, and a third intermediate populated only under continuous illumination conditions. Our data provide novel insight into the photoactive site of channelrhodopsin-2 during the photocycle. They further show that DNP-enhanced solid-state NMR fills the gap for challenging membrane proteins between functional studies and X-ray–based structure analysis, which is required for resolving molecular mechanisms.

Wednesday, August 19, 2015

Room temperature hyperpolarization of nuclear spins in bulk


Tateishi, K., et al., Room temperature hyperpolarization of nuclear spins in bulk. Proc Natl Acad Sci U S A, 2014. 111(21): p. 7527-30.


Dynamic nuclear polarization (DNP), a means of transferring spin polarization from electrons to nuclei, can enhance the nuclear spin polarization (hence the NMR sensitivity) in bulk materials at most 660 times for (1)H spins, using electron spins in thermal equilibrium as polarizing agents. By using electron spins in photo-excited triplet states instead, DNP can overcome the above limit. We demonstrate a (1)H spin polarization of 34%, which gives an enhancement factor of 250,000 in 0.40 T, while maintaining a bulk sample ( approximately 0.6 mg, approximately 0.7 x 0.7 x 1 mm(3)) containing >10(19) (1)H spins at room temperature. Room temperature hyperpolarization achieved with DNP using photo-excited triplet electrons has potentials to be applied to a wide range of fields, including NMR spectroscopy and MRI as well as fundamental physics.

Tuesday, August 18, 2015

[NMR] PhD position in Duesseldorf (Germany)

From the Ampere Magnetic Resonance List:



A PhD positions position is available in the field of NMR characterization of membrane protein structure and function. The positions will be part of the research group of Manuel Etzkorn at the University of Duesseldorf, Germany. 

The group focuses on the characterization of pharmacological important membrane proteins as well as the development of novel NMR techniques tailored for these challenging systems. Depending on individual qualification and interest you will have the opportunity to work on various aspects of this highly interdisciplinary topic including (cell-free) protein expression, biophysical assays, numerical simulations as well as solution- and solid-state NMR spectroscopy. 

Candidates should be highly motivated with a strong interest in biophysics. Previous experience in at least one of the following areas: protein biochemistry, biophysical characterization of (membrane) proteins and/or NMR spectroscopy is required. Funding for PhD students is guaranteed for 3 years starting October 2015 or later.

The biomolecular NMR center of the University of Duesseldorf as well as a close collaboration with the Helmholtz Research Center (Forschungszentrum Jülich) offers an exciting and stimulating research environment. Access to state-of-the art instrumentation including high-field solution- and solid-state NMR spectrometers operating at 900 MHz, 800 MHz, 700 MHz, 3x 600 MHz, 600 MHz DNP is available. Additional spectrometers (800 MHz, 800 MHz DNP, 1200 MHz) will become available in/around 2016. 

Düsseldorf is a very pleasant mid-size German city with a vivid historic center located directly at the scenic Rhein-river and offers a large variety of cultural and recreational activities.

Applications should include a cover letter with a brief description of previous research experience, a CV, and contact information for at least two reference persons (typically including the supervisors of the master thesis). Applications and informal inquiries about the lab and research projects should be directed by email to Manuel Etzkorn (manuel.etzkorn@hhu.de).

Links:
Institute homepage: 
Institute of complex systems at the Helmholtz Research Centre Jülich:
Group homepage:

Please feel free to forward this message to potential candidates.
————————————————————————————-----------
Dr. Manuel Etzkorn

Institute of Physical Biology
Heinrich Heine University
Universitätsstr. 1
Bldg. 26.12, U1, room 25
40225 Duesseldorf

Phone: +49-(0)211 81 12023

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Monday, August 17, 2015

Hybrid polarizing solids for pure hyperpolarized liquids through dissolution dynamic nuclear polarization


Gajan, D., et al., Hybrid polarizing solids for pure hyperpolarized liquids through dissolution dynamic nuclear polarization. Proc. Nat. Aca. Sci. USA, 2014. 111(41): p. 14693-14697.


Hyperpolarization of substrates for magnetic resonance spectroscopy (MRS) and imaging (MRI) by dissolution dynamic nuclear polarization (D-DNP) usually involves saturating the ESR transitions of polarizing agents (PAs; e.g., persistent radicals embedded in frozen glassy matrices). This approach has shown enormous potential to achieve greatly enhanced nuclear spin polarization, but the presence of PAs and/or glassing agents in the sample after dissolution can raise concerns for in vivo MRI applications, such as perturbing molecular interactions, and may induce the erosion of hyperpolarization in spectroscopy and MRI. We show that D-DNP can be performed efficiently with hybrid polarizing solids (HYPSOs) with 2,2,6,6-tetramethyl-piperidine-1-oxyl radicals incorporated in a mesostructured silica material and homogeneously distributed along its pore channels. The powder is wetted with a solution containing molecules of interest (for example, metabolites for MRS or MRI) to fill the pore channels (incipient wetness impregnation), and DNP is performed at low temperatures in a very efficient manner. This approach allows high polarization without the need for glass-forming agents and is applicable to a broad range of substrates, including peptides and metabolites. During dissolution, HYPSO is physically retained by simple filtration in the cryostat of the DNP polarizer, and a pure hyperpolarized solution is collected within a few seconds. The resulting solution contains the pure substrate, is free from any paramagnetic or other pollutants, and is ready for in vivo infusion.

Friday, August 14, 2015

Visualizing Specific Cross-Protomer Interactions in the Homo-Oligomeric Membrane Protein Proteorhodopsin by Dynamic-Nuclear-Polarization-Enhanced Solid-State NMR


Maciejko, J., et al., Visualizing Specific Cross-Protomer Interactions in the Homo-Oligomeric Membrane Protein Proteorhodopsin by Dynamic-Nuclear-Polarization-Enhanced Solid-State NMR. J Am Chem Soc, 2015. 137(28): p. 9032-43.


Membrane proteins often form oligomeric complexes within the lipid bilayer, but factors controlling their assembly are hard to predict and experimentally difficult to determine. An understanding of protein-protein interactions within the lipid bilayer is however required in order to elucidate the role of oligomerization for their functional mechanism and stabilization. Here, we demonstrate for the pentameric, heptahelical membrane protein green proteorhodopsin that solid-state NMR could identify specific interactions at the protomer interfaces, if the sensitivity is enhanced by dynamic nuclear polarization. For this purpose, differently labeled protomers have been assembled into the full pentamer complex embedded within the lipid bilayer. We show for this proof of concept that one specific salt bridge determines the formation of pentamers or hexamers. Data are supported by laser-induced liquid bead ion desorption mass spectrometry and by blue native polyacrylamide gel electrophoresis analysis. The presented approach is universally applicable and opens the door toward analyzing membrane protein interactions within homo-oligomers directly in the membrane.

Wednesday, August 12, 2015

NMR Signal Enhancement by Effective SABRE Labeling of Oligopeptides


Ratajczyk, T., et al., NMR Signal Enhancement by Effective SABRE Labeling of Oligopeptides. Chemistry, 2015: p. n/a-n/a.


Signal amplification by reversible exchange (SABRE) can enhance nuclear magnetic resonance signals by several orders of magnitude. However, until now this was limited to a small number of model target molecules. Here, a new convenient method for SABRE activation applicable to a variety of synthetic model oligopeptides is demonstrated. For the first time, a highly SABRE-active pyridine-based biocompatible molecular framework is incorporated into synthetic oligopeptides. The SABRE activity is preserved, demonstrating the importance of such earmarking. Finally, a crucial exchange process responsible for SABRE activity is identified and discussed.

Monday, August 10, 2015

Biomolecular DNP-Supported NMR Spectroscopy using Site-Directed Spin Labeling


van der Cruijsen, E.A.W., et al., Biomolecular DNP-Supported NMR Spectroscopy using Site-Directed Spin Labeling. Chemistry – A European Journal, 2015: p. n/a-n/a.


Dynamic nuclear polarization (DNP) has been shown to greatly enhance spectroscopic sensitivity, creating novel opportunities for NMR studies on complex and large molecular assemblies in life and material sciences. In such applications, however, site-specificity and spectroscopic resolution become critical factors that are usually difficult to control by current DNP-based approaches. We have examined in detail the effect of directly attaching mono- or biradicals to induce local paramagnetic relaxation effects and, at the same time, to produce sizable DNP enhancements. Using a membrane-embedded ion channel as an example, we varied the degree of paramagnetic labeling and the location of the DNP probes. Our results show that the creation of local spin clusters can generate sizable DNP enhancements while preserving the intrinsic benefits of paramagnetic relaxation enhancement (PRE)-based NMR approaches. DNP using chemical labeling may hence provide an attractive route to introduce molecular specificity into DNP studies in life science applications and beyond.

Friday, August 7, 2015

Design of a hyperpolarized 15N NMR probe that induces a large chemical-shift change upon binding of calcium ions


Hata, R., et al., Design of a hyperpolarized 15N NMR probe that induces a large chemical-shift change upon binding of calcium ions. Chemical Communications, 2015. 51(61): p. 12290-12292.


Ca2+ is a fundamental metal ion for physiological functioning. Therefore, molecular probes for Ca2+ analysis are required. Recently, a hyperpolarized NMR probe has emerged as a promising tool. Here, we report a new design of a hyperpolarized NMR probe for Ca2+, which showed a large chemical shift change upon binding to Ca2+ and was applied for Ca2+ sensing in a hyperpolarized state.

Thursday, August 6, 2015

[NMR] Employment opportunity: NMR Facility Manager at the University of Guelph, Ontario, Canada

From the Ampere Magnetic Resonance List:



Dear Colleagues,

The NMR Centre at the University of Guelph, ON, Canada, has an opening for a Facility Manager. Please forward to anyone who might be interested. 

Many thanks, 
Vlad Ladizhansky


NMR Centre Manager
NMR Centre, College of Physical and Engineering Science

Reporting to the Director of the University of Guelph NMR Centre, the NMR Centre Manager will oversee all aspects of the operation of the Centre which includes six Bruker NMR spectrometers operating at 800, 600 (2), 500, 400, and 300 MHz capable of both solution and solid-state NMR work, and a 600 MHz/395 GHz Dynamic Nuclear Polarization solid-state NMR spectrometer. The NMR spectrometers are equipped with a large number of solution and solid state NMR probes including the 600 MHz cryoprobe and a 400 MHz Prodigy probe. The Manager will be responsible for: the development and provision of training procedures, and assisting internal and external users with a wide range of research needs; the supervision of full- and part-time support staff employed by the Centre; training new NMR spectrometer users; ongoing involvement in collaborative research efforts with faculty including writing research proposals for operations and new equipment acquisitions; preparing and overseeing the Centre’s budget, which includes revenues from major and minor users, as well as from industrial and government clients; consulting with clients to solve problems; supervising the resolution of ongoing issues with instrument/ experimental problems; and safety in the laboratory, ensuring procedures are followed.

Requirements of the position include: Ph.D. in a relevant area of biochemistry, biophysics or chemistry with six years of related experience or equivalent combination of education and experience. Additional requirements include: demonstrated expertise in NMR spectroscopy; a thorough knowledge of modern NMR methods and applications, hardware and software; ability to implement the newest experimental developments in NMR spectroscopy; experience in troubleshooting NMR equipment and in handling cryogens; supervisory skills including effective teambuilding and the ability to establish positive working relationships and foster collaboration to achieve common goals; experience in training and monitoring performance; strong verbal and written communication skills; strong organizational, decision-making and management skills.

Hiring #: 2015-0226

Classification P07*
*Tentative evaluation; subject to committee review

For more information on the position, professional and managerial salary bands, and how to apply, please visit:

Dr. Vladimir Ladizhansky
Associate Professor
Department of Physics 
University of Guelph, 
50 Stone Road E, Guelph, 
Ontario, Canada N1G 2W1
phone: 519-824-4120 Ext 53989
departmental fax: 519-836-9967 

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Wednesday, August 5, 2015

Matrix-free DNP-enhanced NMR spectroscopy of liposomes using a lipid-anchored biradical


Fernandez-de-Alba, C., et al., Matrix-free DNP-enhanced NMR spectroscopy of liposomes using a lipid-anchored biradical. Chemistry, 2015. 21(12): p. 4512-7.


Magic-angle spinning dynamic nuclear polarization (MAS-DNP) has been proven to be a powerful technique to enhance the sensitivity of solid-state NMR (SSNMR) in a wide range of systems. Here, we show that DNP can be used to polarize lipids using a lipid-anchored polarizing agent. More specifically, we introduce a C16-functionalized biradical, which allows localization of the polarizing agents in the lipid bilayer and DNP experiments to be performed in the absence of excess cryo-protectant molecules (glycerol, dimethyl sulfoxide, etc.). This constitutes another original example of the matrix-free DNP approach that we recently introduced.

Monday, August 3, 2015

Solid-state NMR methods for oriented membrane proteins


This review article gives a comprehensive overview of the field of solid-state NMR spectroscopy on oriented samples. These experiments are typically less demanding on instrumentation since experiments are performed on static samples, without the need of spinning. While this article is not directly related to DNP-NMR spectroscopy, DNP-NMR on static samples will certainly become more interesting in the future.


Hansen, S.K., et al., Solid-state NMR methods for oriented membrane proteins. Prog. NMR. Spec., 2015. 88-89: p. 48-85.


Oriented-sample solid-state NMR represents one of few experimental methods capable of characterising the membrane-bound conformation of proteins in the cell membrane. Since the technique was developed 25 years ago, the technique has been applied to study the structure of helix bundle membrane proteins and antimicrobial peptides, characterise protein-lipid interactions, and derive information on dynamics of the membrane anchoring of membrane proteins. We will review the major developments in various aspects of oriented-sample solid-state NMR, including sample-preparation methods, pulse sequences, theory required to interpret the experiments, perspectives for and guidelines to new experiments, and a number of representative applications.