FMP Publications

Our publications are recorded in a searchable database since 2010, updates will be added regularly.

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References
High-Temperature Dynamic Nuclear Polarization Enhanced Magic-Angle-Spinning NMR
Akbey, Ü., Linden, A. H.; Oschkinat, H.
Appl Magn Reson, 43:81-90
(2012)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Dynamic nuclear polarization (DNP) transfers electron spin-polarization to nuclear spins in close proximity, increasing sensitivity by two-to-three orders of magnitude. This enables nuclear magnetic resonance (NMR) experiments on samples with low concentrations of analyte. The requirement of using cryogenic temperatures in DNP-enhanced solid-state NMR (ssNMR) experiments may impair the resolution and hence limit its broad application to biological systems. In this work, we introduce a "High-Temperature DNP" approach, which aims at increasing spectral resolution by performing experiments at temperatures of around 180 K instead of similar to 100 K. By utilizing the extraordinary enhancements obtained on deuterated proteins, still sufficiently large DNP enhancements of 11-18 are obtained for proton and carbon, respectively. We recorded high sensitivity 2D C-13-C-13 spectra in similar to 9 min with higher resolution than at 100 K, which has similar resolution to the one obtained at room temperature for some favorable residues.

Practical aspects of high-sensitivity multidimensional (1)(3)C MAS NMR spectroscopy of perdeuterated proteins
Akbey, Ü., Rossum, B. J.; Oschkinat, H.
J Magn Reson, 217:77-85
(2012)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: The double nucleus enhanced recoupling (DONER) experiment employs simultaneous irradiation of protons and deuterons to promote spin diffusion processes in a perdeuterated protein. This results in 4-5 times higher sensitivity in 2D (13)C-(13)C correlation experiments as compared to PDSD [1]. Here, a quantitative comparison of PDSD, (1)H-DARR, (2)H-DARR, and (1)H+(2)H DONER has been performed to analyze the influence of spin diffusion on polarization transfer processes. Cross peak buildup curves were analyzed to obtain guidelines for choosing the best experimental parameters. The largest cross peak intensities were observed for the DONER experiments. The fastest build-up rate was observed in the (2)H-DARR experiment within a buildup range of approximately 18-45 ms, whereas values between 24 and 69 ms are observed for the DONER experiment. Furthermore, the effects of direct excitation and cross polarization (CP) are compared. A comparison between DONER and RFDR experiments reveal approximately 50% more intense cross peaks in the C(alpha)-CO and C(alpha)-C(alip) regions of the 2D (13)C-(13)C DONER spectrum applying proton CP ((1)H-(13)C). As a parameter determining the S/N in (13)C-(13)C correlation experiments, proton CP efficiency is investigated using deuterated samples with proton/deuterium ratios at 20%, 40%, and 100% H(2)O. Sufficiently strong (13)C CPMAS signal intensity is observed for such proteins even with very low proton concentration. The effect of proton and/or deuterium decoupling is analyzed at various MAS spinning frequencies. Deuterium decoupling was found most crucial for obtaining high resolution. Long range correlations are readily observed representing distances up to approximately 6 A by using DONER approach.

Efficient Modeling of Symmetric Protein Aggregates from NMR Data
Bardiaux, B., van Rossum, B. J., Nilges(*), M.; Oschkinat, H.
Angew Chem Int Edit, 51:6916-6919
(2012)

Tags: NMR-Supported Structural Biology (Oschkinat)

Rapid solid-state NMR of deuterated proteins by interleaved cross-polarization from H-1 and H-2 nuclei
Bjerring(*), M., Paaske(*), B., Oschkinat, H., Akbey, Ü.; Nielsen(*), N. C.
Journal of Magnetic Resonance, 214:324-328
(2012)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: We present a novel sampling strategy, interleaving acquisition of multiple NMR spectra by exploiting initial polarization subsequently from H-1 and H-2 spins, taking advantage of their different T-1 relaxation times. Different H-1- and H-2-polarization based spectra are in this way simultaneously recorded improving either information content or sensitivity by adding spectra. The so-called Relaxation-optimized Acquisition of Proton Interleaved with Deuterium (RAPID) H-1 -> C-13/H-2 -> C-13 CP/MAS multiple-acquisition method is demonstrated by 1D and 2D experiments using a uniformly H-2, N-15, C-13-labeled alpha-spectrin SH3 domain sample with all or 30% back-exchanged labile H-2 to H-1. It is demonstrated how 1D C-13 CP/MAS or 2D C-13-C-13 correlation spectra initialized with polarization from either H-1 or H-2 may be recorded simultaneously with flexibility to be added or used individually for spectral editing. It is also shown how 2D C-13-C-13 correlation spectra may be recorded interleaved with H-2-C-13 correlation spectra to obtain C-13-C-13 correlations along with information about dynamics from H-2 sideband patterns. (C) 2011 Elsevier Inc. All rights reserved.

Solid-state magic-angle spinning NMR of membrane proteins and protein-ligand interactions
Franks, W. T., Linden, A. H., Kunert, B., van Rossum, B. J.; Oschkinat, H.
Eur J Cell Biol, 91:340-348
(2012)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Structural biology is developing into a universal tool for visualizing biological processes in space and time at atomic resolution. The field has been built by established methodology like X-ray crystallography, electron microscopy and solution NMR and is now incorporating new techniques, such as small-angle X-ray scattering, electron tomography, magic-angle-spinning solid-state NMR and femtosecond X-ray protein nanocrystallography. These new techniques all seek to investigate non-crystalline, native-like biological material. Solid-state NMR is a relatively young technique that has just proven its capabilities for de novo structure determination of model proteins. Further developments promise great potential for investigations on functional biological systems such as membrane-integrated receptors and channels, and macromolecular complexes attached to cytoskeletal proteins. Here, we review the development and applications of solid-state NMR from the first proof-of-principle investigations to mature structure determination projects, including membrane proteins. We describe the development of the methodology by looking at examples in detail and provide an outlook towards future 'big' projects.

Characterization of Membrane Proteins in Isolated Native Cellular Membranes by Dynamic Nuclear Polarization Solid-State NMR Spectroscopy without Purification and Reconstitution
Jacso, T., Franks, W. T., Rose, H., Fink, U., Broecker(*), J., Keller(*), S., Oschkinat, H.; Reif, B.
Angew Chem Int Edit, 51:432-435
(2012)

Tags: Solid-State NMR Spectroscopy (Reif)

The effect of biradical concentration on the performance of DNP-MAS-NMR
Lange, S., Linden, A. H., Akbey, Ü., Franks, W. T., Loening(*), N. M., van Rossum, B. J.; Oschkinat, H.
J Magn Reson, 216:209-212
(2012)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: With the technique of dynamic nuclear polarization (DNP) signal intensity in solid-state MAS-NMR experiments can be enhanced by 2-3 orders of magnitude. DNP relies on the transfer of electron spin polarization from unpaired electrons to nuclear spins. For this reason, stable organic biradicals such as TOTAPOL are commonly added to samples used in DNP experiments. We investigated the effects of biradical concentration on the relaxation, enhancement, and intensity of NMR signals, employing a series of samples with various TOTAPOL concentrations and uniformly (13)C, (15)N labeled proline. A considerable decrease of the NMR relaxation times (T(1), T(2)( *), and T(1)(rho)) is observed with increasing amounts of biradical due to paramagnetic relaxation enhancement (PRE). For nuclei in close proximity to the radical, decreasing T(1)(rho) reduces cross-polarization efficiency and decreases in T(2)( *) broaden the signal. Additionally, paramagnetic shifts of (1)H signals can cause further line broadening by impairing decoupling. On average, the combination of these paramagnetic effects (PE; relaxation enhancement, paramagnetic shifts) quenches NMR-signals from nuclei closer than 10A to the biradical centers. On the other hand, shorter T(1) times allow the repetition rate of the experiment to be increased, which can partially compensate for intensity loss. Therefore, it is desirable to optimize the radical concentration to prevent additional line broadening and to maximize the signal-to-noise observed per unit time for the signals of interest.

A comparison of NCO and NCA transfer methods for biological solid-state NMR spectroscopy
Loening, N. M., Bjerring(*), M., Nielsen(*), N. C.; Oschkinat, H.
Journal of Magnetic Resonance, 214:81-90
(2012)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Three different techniques (adiabatic passage Hartman-Hahn cross-polarization, optimal control designed pulses, and EXPORT) are compared for transferring N-15 magnetization to C-13 in solid-state NMR experiments under magic-angle-spinning conditions. We demonstrate that, in comparison to adiabatic passage Hartman-Hahn cross-polarization, optimal control transfer pulses achieve similar or better transfer efficiencies for uniformly-C-13,N-15 labeled samples and are generally superior for samples with non-uniform labeling schemes (such as 1,3- and 2-C-13 glycerol labeling). In addition, the optimal control pulses typically use substantially lower average RF field strengths and are more robust with respect to experimental variation and RF inhomogeneity. Consequently, they are better suited for demanding samples. (C) 2011 Elsevier Inc. All rights reserved.

Broadband excitation pulses for high-field solid-state nuclear magnetic resonance spectroscopy
Loening, N. M., van Rossum, B. J.; Oschkinat, H.
Magn. Reson. Chem., 50:284-288
(2012)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: In nuclear magnetic resonance spectroscopy, experimental limits due to the radiofrequency transmitter and/or coil means that conventional radiofrequency pulses (hard pulses) are sometimes not sufficiently powerful to excite magnetization uniformly over a desired range of frequencies. Effects due to nonuniform excitation are most frequently encountered at high magnetic fields for nuclei with a large range of chemical shifts. Using optimal control theory, we have designed broadband excitation pulses that are suitable for solid-state samples under magic-angle-spinning conditions. These pulses are easy to implement, robust to spinning frequency variations, and radiofrequency inhomogeneities, and only four times as long as a corresponding hard pulse. The utility of these pulses for uniformly exciting 13C nuclei is demonstrated on a 900 MHz (21.1 T) spectrometer. Copyright (c) 2012 John Wiley & Sons, Ltd.

Fast passage dynamic nuclear polarization on rotating solids
Mentink-Vigier(*), F., Akbey, Ü., Hovav(*), Y., Vega(*), S., Oschkinat, H.; Feintuch(*), A.
Journal of Magnetic Resonance, 224:13-21
(2012)

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Magic Angle Spinning (MAS) Dynamic Nuclear Polarization (DNP) has proven to be a very powerful way to improve the signal to noise ratio of NMR experiments on solids. The experiments have in general been interpreted considering the Solid-Effect (SE) and Cross-Effect (CE) DNP mechanisms while ignoring the influence of sample spinning. In this paper, we show experimental data of MAS-DNP enhancements of H-1 and C-13 in proline and SH3 protein in glass forming water/glycerol solvent containing TOTAPOL. We also introduce a theoretical model that aims at explaining how the nuclear polarization is built in MAS-DNP experiments. By using Liouville space based simulations to include relaxation on two simple spin models, (electron-nucleus) and (electron-electron-nucleus), we explain how the basic MAS-SE-DNP and MAS-CE-DNP processes work. The importance of fast energy passages and short level anti-crossing is emphasized and the differences between static DNP and MAS-DNP is explained. During a single rotor cycle the enhancement in the (electron-electron-nucleus) system arises from MAS-CE-DNP involving at least three kinds of two-level fast passages: an electron-electron dipolar anti-crossing, a single quantum electron MW encounter and an anti-crossing at the CE condition inducing nuclear polarization in- or decrements. Numerical, powder-averaged, simulations were performed in order to check the influence of the experimental parameters on the enhancement efficiencies. In particular we show that the spinning frequency dependence of the theoretical MAS-CE-DNP enhancement compares favorably with the experimental and 13C MAS-DNP enhancements of proline and SH3. (C) 2012 Elsevier Inc. All rights reserved.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
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