FMP Publications

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

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Structural biology applications of solid state MAS DNP NMR
Akbey(*), Ü.; Oschkinat, H.
J Magn Reson, 269:213-224

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Dynamic Nuclear Polarization (DNP) has long been an aim for increasing sensitivity of nuclear magnetic resonance (NMR) spectroscopy, delivering spectra in shorter experiment times or of smaller sample amounts. In recent years, it has been applied in magic angle spinning (MAS) solid-state NMR to a large range of samples, including biological macromolecules and functional materials. New research directions in structural biology can be envisaged by DNP, facilitating investigations on very large complexes or very heterogeneous samples. Here we present a summary of state of the art DNP MAS NMR spectroscopy and its applications to structural biology, discussing the technical challenges and factors affecting DNP performance.

On The Potential of Dynamic Nuclear Polarization Enhanced Diamonds in Solid-State and Dissolution (13) C NMR Spectroscopy
Bretschneider(*), C. O., Akbey, Ü., Aussenac(*), F., Olsen(*), G. L., Feintuch(*), A., Oschkinat, H.; Frydman(*), L.
Chemphyschem, 17:2691-2701

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Dynamic nuclear polarization (DNP) is a versatile option to improve the sensitivity of NMR and MRI. This versatility has elicited interest for overcoming potential limitations of these techniques, including the achievement of solid-state polarization enhancement at ambient conditions, and the maximization of (13) C signal lifetimes for performing in vivo MRI scans. This study explores whether diamond's (13) C behavior in nano- and micro-particles could be used to achieve these ends. The characteristics of diamond's DNP enhancement were analyzed for different magnetic fields, grain sizes, and sample environments ranging from cryogenic to ambient temperatures, in both solution and solid-state experiments. It was found that (13) C NMR signals could be boosted by orders of magnitude in either low- or room-temperature solid-state DNP experiments by utilizing naturally occurring paramagnetic P1 substitutional nitrogen defects. We attribute this behavior to the unusually long electronic/nuclear spin-lattice relaxation times characteristic of diamond, coupled with a time-independent cross-effect-like polarization transfer mechanism facilitated by a matching of the nitrogen-related hyperfine coupling and the (13) C Zeeman splitting. The efficiency of this solid-state polarization process, however, is harder to exploit in dissolution DNP-enhanced MRI contexts. The prospects for utilizing polarized diamond approaching nanoscale dimensions for both solid and solution applications are briefly discussed.

Chemical shift assignments and secondary structure prediction for Q4DY78, a conserved kinetoplastid-specific protein from Trypanosoma cruzi
D'Andrea(*), E. D., Diehl, A., Schmieder, P., Oschkinat, H.; Pires(*), J. R.
Biomol NMR Assign, 10:325-328

Tags: NMR-Supported Structural Biology (Oschkinat), Solution NMR (Schmieder)

Abstract: Trypanosoma cruzi, Trypanosma brucei and Leishmania spp. are kinetoplastid protozoa causative agents of Chagas disease, sleeping sickness and leishmaniasis, respectively, neglected tropical diseases estimated to infect millions of people worldwide. Their genome sequencing has revealed approximately 50 % of genes encoding hypothetical proteins of unknown function, opening possibilities for novel target identification and drug discovery. Q4DY78 is a putative essential protein from T. cruzi conserved in the related kinetoplastids and divergent from mammalian host proteins. Here we report the (1)H, (15)N, and (13)C chemical shift assignments and secondary structure analysis of the Q4DY78 protein as basis for NMR structure determination, functional analysis and drug screening.

Structural basis for the dissociation of alpha-synuclein fibrils triggered by pressure perturbation of the hydrophobic core
de Oliveira(*), G. A., Marques(*), M. A., Cruzeiro-Silva(*), C., Cordeiro(*), Y., Schuabb(*), C., Moraes(*), A. H., Winter(*), R., Oschkinat, H., Foguel(*), D., Freitas(*), M. S.; Silva(*), J. L.
Sci Rep, 6:37990

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Parkinson's disease is a neurological disease in which aggregated forms of the alpha-synuclein (alpha-syn) protein are found. We used high hydrostatic pressure (HHP) coupled with NMR spectroscopy to study the dissociation of alpha-syn fibril into monomers and evaluate their structural and dynamic properties. Different dynamic properties in the non-amyloid-beta component (NAC), which constitutes the Greek-key hydrophobic core, and in the acidic C-terminal region of the protein were identified by HHP NMR spectroscopy. In addition, solid-state NMR revealed subtle differences in the HHP-disturbed fibril core, providing clues to how these species contribute to seeding alpha-syn aggregation. These findings show how pressure can populate so far undetected alpha-syn species, and they lay out a roadmap for fibril dissociation via pathways not previously observed using other approaches. Pressure perturbs the cavity-prone hydrophobic core of the fibrils by pushing water inward, thereby inducing the dissociation into monomers. Our study offers the molecular details of how hydrophobic interaction and the formation of water-excluded cavities jointly contribute to the assembly and stabilization of the fibrils. Understanding the molecular forces behind the formation of pathogenic fibrils uncovered by pressure perturbation will aid in the development of new therapeutics against Parkinson's disease.

Temperature dependence of cross-effect dynamic nuclear polarization in rotating solids: advantages of elevated temperatures
Geiger, M. A., Orwick-Rydmark, M., Marker, K., Franks, W. T., Akhmetzyanov(*), D., Stöppler, D., Zinke, M., Specker, E., Nazare, M., Diehl, A., van Rossum, B. J., Aussenac(*), F., Prisner(*), T., Akbey, Ü.; Oschkinat, H.
Phys Chem Chem Phys, 18:30696-30704

Tags: NMR-Supported Structural Biology (Oschkinat), Medicinal Chemistry (Nazare), Molecular Biophysics (Lange, A.)

Abstract: Dynamic nuclear polarization exploits electron spin polarization to boost signal-to-noise in magic-angle-spinning (MAS) NMR, creating new opportunities in materials science, structural biology, and metabolomics studies. Since protein NMR spectra recorded under DNP conditions can show improved spectral resolution at 180-200 K compared to 110 K, we investigate the effects of AMUPol and various deuterated TOTAPOL isotopologues on sensitivity and spectral resolution at these temperatures, using proline and reproducibly prepared SH3 domain samples. The TOTAPOL deuteration pattern is optimized for protein DNP MAS NMR, and signal-to-noise per unit time measurements demonstrate the high value of TOTAPOL isotopologues for Protein DNP MAS NMR at 180-200 K. The combined effects of enhancement, depolarization, and proton longitudinal relaxation are surprisingly sample-specific. At 200 K, DNP on SH3 domain standard samples yields a 15-fold increase in signal-to-noise over a sample without radicals. 2D and 3D NCACX/NCOCX spectra were recorded at 200 K within 1 and 13 hours, respectively. Decreasing enhancements with increasing 2H-content at the CH2 sites of the TEMPO rings in CD3-TOTAPOL highlight the importance of protons in a sphere of 4-6 A around the nitroxyl group, presumably for polarization pickup from electron spins.

Studying the Conformation of a Silaffin-Derived Pentalysine Peptide Embedded in Bioinspired Silica using Solution and Dynamic Nuclear Polarization Magic-Angle Spinning NMR
Geiger(*), Y., Gottlieb(*), H. E., Akbey, Ü., Oschkinat, H.; Goobes(*), G.
J Am Chem Soc, 138:5561-5567

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Smart materials are created in nature at interfaces between biomolecules and solid materials. The ability to probe the structure of functional peptides that engineer biogenic materials at this heterogeneous setting can be facilitated tremendously by use of DNP-enhanced solid-state NMR spectroscopy. This sensitive NMR technique allows simple and quick measurements, often without the need for isotope enrichment. Here, it is used to characterize a pentalysine peptide, derived from a diatom's silaffin protein. The peptide accelerates the formation of bioinspired silica and gets embedded inside the material as it is formed. Two-dimensional DNP MAS NMR of the silica-bound peptide and solution NMR of the free peptide are used to derive its secondary structure in the two states and to pinpoint some subtle conformational changes that the peptide undergoes in order to adapt to the silica environment. In addition, interactions between abundant lysine residues and silica surface are identified, and proximity of other side chains to silica and to neighboring peptide molecules is discussed.

Dynamic Nuclear Polarization Enhanced MAS NMR Spectroscopy for Structural Analysis of HIV-1 Protein Assemblies
Gupta(*), R., Lu(*), M., Hou(*), G., Caporini(*), M. A., Rosay(*), M., Maas(*), W., Struppe(*), J., Suiter(*), C., Ahn(*), J., Byeon(*), I. J., Franks, W. T., Orwick-Rydmark, M., Bertarello(*), A., Oschkinat, H., Lesage(*), A., Pintacuda(*), G., Gronenborn(*), A. M.; Polenova(*), T.
J Phys Chem B, 120:329-339

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Mature infectious HIV-1 virions contain conical capsids composed of CA protein, generated by the proteolytic cleavage cascade of the Gag polyprotein, termed maturation. The mechanism of capsid core formation through the maturation process remains poorly understood. We present DNP-enhanced MAS NMR studies of tubular assemblies of CA and Gag CA-SP1 maturation intermediate and report 20-64-fold sensitivity enhancements due to DNP at 14.1 T. These sensitivity enhancements enabled direct observation of spacer peptide 1 (SP1) resonances in CA-SP1 by dipolar-based correlation experiments, unequivocally indicating that the SP1 peptide is unstructured in assembled CA-SP1 at cryogenic temperatures, corroborating our earlier results. Furthermore, the dependence of DNP enhancements and spectral resolution on magnetic field strength (9.4-18.8 T) and temperature (109-180 K) was investigated. Our results suggest that DNP-based measurements could potentially provide residue-specific dynamics information by allowing for the extraction of the temperature dependence of the anisotropic tensorial or relaxation parameters. With DNP, we were able to detect multiple well-resolved isoleucine side-chain conformers; unique intermolecular correlations across two CA molecules; and functionally relevant conformationally disordered states such as the 14-residue SP1 peptide, none of which are visible at ambient temperatures. The detection of isolated conformers and intermolecular correlations can provide crucial constraints for structure determination of these assemblies. Overall, our results establish DNP-based MAS NMR spectroscopy as an excellent tool for the characterization of HIV-1 assemblies.

bcTol : a highly water-soluble biradical for efficient dynamic nuclear polarization of biomolecules
Jagtap(*), A. P., Geiger, M. A., Stöppler, D., Orwick-Rydmark, M., Oschkinat, H.; Sigurdsson(*), S. T.
Chem Commun (Camb), 52:7020-7023

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: Dynamic nuclear polarization (DNP) is an efficient method to overcome the inherent low sensitivity of magic-angle spinning (MAS) solid-state NMR. We report a new polarizing agent (), designed for biological applications, that yielded an enhancement value of 244 in a microcrystalline SH3 domain sample at 110 K.

Structural analysis of a signal peptide inside the ribosome tunnel by DNP MAS NMR
Lange, S., Franks, W. T., Rajagopalan(*), N., Döring(*), K., Geiger, M. A., Linden, A., van Rossum, B. J., Kramer(*), G., Bukau(*), B.; Oschkinat, H.
Sci Adv, 2:e1600379

Tags: NMR-Supported Structural Biology (Oschkinat), Molecular Biophysics (Lange, A.)

Abstract: Proteins are synthesized in cells by ribosomes and, in parallel, prepared for folding or targeting. While ribosomal protein synthesis is progressing, the nascent chain exposes amino-terminal signal sequences or transmembrane domains that mediate interactions with specific interaction partners, such as the signal recognition particle (SRP), the SecA-adenosine triphosphatase, or the trigger factor. These binding events can set the course for folding in the cytoplasm and translocation across or insertion into membranes. A distinction of the respective pathways depends largely on the hydrophobicity of the recognition sequence. Hydrophobic transmembrane domains stabilize SRP binding, whereas less hydrophobic signal sequences, typical for periplasmic and outer membrane proteins, stimulate SecA binding and disfavor SRP interactions. In this context, the formation of helical structures of signal peptides within the ribosome was considered to be an important factor. We applied dynamic nuclear polarization magic-angle spinning nuclear magnetic resonance to investigate the conformational states of the disulfide oxidoreductase A (DsbA) signal peptide stalled within the exit tunnel of the ribosome. Our results suggest that the nascent chain comprising the DsbA signal sequence adopts an extended structure in the ribosome with only minor populations of helical structure.

Quantitative and Qualitative Analysis of Surface Modified Cellulose Utilizing TGA-MS
Loof(*), D., Hiller, M., Oschkinat, H.; Koschek(*), K.
Materials (Basel, Switzerland), 9

Tags: NMR-Supported Structural Biology (Oschkinat)

Abstract: With the aim to enhance interfacial adhesion of a hydrophobic polymer matrix and cellulosic fibers and fillers, chemical surface modifications with silane coupling agents are performed. Thermogravimetric analysis (TGA) could be used to determine the degree of surface functionalization. However, similar thermal properties of treated and untreated cellulose hamper a precise determination of silane loading. This contribution deals with quantitative determination of silane loading combining both TGA and elemental analysis. Firstly, silane modified celluloses were studied by FT-IR, Raman, solid state NMR spectroscopy, and polarized light microscopy in order to determine functional groups and to study the impact of chemical treatment on cellulose morphology. Secondly, thermal stability and pyrolysis processes were studied by TG-MS analysis. In order to determine the exact silane loading, the mass percentages of the appropriate elements were quantified by elemental analysis and correlated with the charred residues determined by TGA yielding a linear dependency. With that correlation, it was possible to determine silane loadings for additional samples utilizing simple TGA measurements. The main advantage of that approach is that only one calibration is necessary for routine analyses of further samples and TGA-MS coupling gives additional information on thermal stability and pyrolysis routes, simultaneously.

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Leibniz-Forschungsinstitut für Molekulare Pharmakologie im Forschungsverbund Berlin e.V. (FMP)
Campus Berlin-Buch
Robert-Roessle-Str. 10
13125 Berlin, Germany
+4930 94793 - 100 
+4930 94793 - 109 (Fax)

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