Dynamic Nuclear Polarization Magic Angle Spinning Nuclear Magnetic Resonance: Method Development and Applications

The signal-to-noise ratio of MAS NMR signals can be enhanced via dynamic nuclear polarization (DNP) by several orders of magnitude. DNP can provide new perspectives in structural biology, metabolomics studies and material science. At cryogenic temperatures (100 K) stable water-soluble nitroxide biradicals are added to the investigated samples as polarizing agents. The large polarization of the biradical electrons can be transferred to the analytes under continuous microwave irradiation via the so-called cross effect (CE). Hence, the use of DNP enables MAS NMR experiments that suffer from inherent low sensitivity and would not have been executed normally due to the excessively long experimental time required for signal averaging. The current challenge in DNP MAS NMR is the homogeneous and heterogeneous line broadening and the temperature dependence of the CE. In this work, experimental conditions, parameters and aspects that determine the CE efficiency were investigated. Novel polarizing agents were tested for the first time, the methodology further developed and the new insights applied to biomolecular projects. The amino acid proline and the 62 amino acid long protein SH3 in microcrystalline form were used as model systems. Four CD3-TOTAPOL isotopologues were investigated and compared to already established biradicals such as TOTAPOL and AMUPol in the course of this work. Their CE efficiency was assessed by comparing enhancement values and the signal-to-noise ratio per 10 min (10minSNR) for 1D hC (1H- 13C CP) experiments in a temperature range between 100 and 200 K at 9.4 T. Signal-to-noise ratio were analyzed and compared with a newly devised procedure. The deuteration of the methyl groups of 1H-TOTAPOL led to larger signal enhancements compared to 1H-TOTAPOL. The effect of deuteration of 1H-TOTAPOL did not influence the electron relaxation parameter as initially hypothesized and are not the reason for increased CE efficiency of the polarizing agent. Highest signal enhancements were obtained for the isotopologue CD3-TOTAPOL-0 that has deuterated methyl groups but protonated 3 and 5 positions of the TEMPO ring. The results highlight the importance of protons that are at close proximity to the radical center, presumably involved in the initial polarization buildup. In addition, the new polarizing agent bcTol, designed for biological applications was tested for the first time in the course of this work. The maximum signal enhancement that could be obtained was e ~ 248 for a sample containing microcrystalline SH3 at 110 K. The bcTol biradical showed unprecedented solubility in water, GDH and [D8]-glycerol. Measurements of signal-to-noise per unit time suggest a comparable DNP performance of bcTol at 110 K to that of AMUPol.Furthermore, a 10minSNR study was conducted to investigate the influence of electron relaxation parameters and the radical linker. Therefore, the novel polarizing agents and cyolyl-TOTAPOL and bcTol-M were investigated for the first time. bcTol-M is similar to bcTol but with methyl groups on the nitrogen atoms showed greater 10minSNR ratio than bcTol. The maximum signal enhancement that could be measured with this radical was e ~ 302 on a proline sample in a 3.2 mm sapphire rotor at 110 K. The superior performance compared to bcTol and AMUPol can be mainly attributed to the shorter 1H-T1 times. The presence of the methyl groups on the linkage presumably promotes nuclear relaxation. Given the simplified handling of the radical and its good performance in hC cross polarization experiments, bcTol-M constitutes an ideal polarizing agent for biomolecular DNP MAS NMR studies. The three urea-based radicals AMUPol, bcTol und bcTol-M show a considerable higher enhancement and SNR compared to 1H-TOTAPol and cyolyl-TOTAPol. Among the urea based radicals, the enhancement values do not differ to a large extent but the values for the SNR do. Furthermore, the electron relaxation times that were measured at the same field as the NMR spectra (9.4 T) for the radicals 1H- TOTAPOL, cyolyl-TOTAPOL, bcTol, AMUPol, and bcTol-M do not dominate the performance of the radicals at 110 K, 8 kHz MAS and 9.4 T. The different performance correlates with the chosen type of linker and the degree of alkylation, affecting the size of the effective electron-electron dipolar coupling and the nuclear relaxation times. A sample preparation protocol for the SH3 samples was developed in order to get reliable and reproducible results. 2D 13C-13C DARR spectra were recorded under DNP conditions for each radical. The best resolution was obtained for the samples containing AMUPol as polarizing agent. The signal-to-noise ratio at 200 K on SH3 standard samples is 15 times larger compared to a sample without polarizing agent. 2D and 3D NCACX/NCOCX spectra were recorded at 200 K within 1 and 13 hours respectively. The obtained resolution allows for some spectral assignment of amino acid side chains. For the novel nitroxide biradical bcTol the signal-to-noise per unit time were higher compared to AMUPol at 181 K. 2D spectra of the SH3 domain sample recorded at 181 K gave a signal enhancement of e ~ 40 and show sufficient resolution for structural studies. In the final sections of this thesis, the power of these new insights and developments were demonstrated on diverse biological problems

bcTol: a highly water-soluble biradical for efficient dynamic nuclear polarization of biomolecules

Post-print (lokagerð höfundar)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 (bcTol), designed for biological applications, that yielded an enhancement value of 244 in a microcrystalline SH3 domain sample at 110 K.This work was financially supported by the Icelandic Research Fund (141062051), the Deutsche Forschungsgemeinschaft (SFB 1078, 740 and 765) and by a doctoral fellowship to A. P. J. from the University of Icelandic Research Fund. We thank A. Diehl, K. Rehbein, N. Erdmann and D. Michl for the preparation of microcrystalline SH3 and channelrhodopsin samples, Dr S. Jonsdottir for assistance in collecting analytical data for structural characterization of the radicals, as well as P. Hegemann and K. Stehfest for helpful discussions concerning expression and purification of channelrhodopsin.Peer reviewe

3D bioprinting of tissue-specific osteoblasts and endothelial cells to model the human jawbone

Jawbone differs from other bones in many aspects, including its developmental origin and the occurrence of jawbone-specific diseases like MRONJ (medication-related osteonecrosis of the jaw). Although there is a strong need, adequate in vitro models of this unique environment are sparse to date. While previous approaches are reliant e.g. on scaffolds or spheroid culture, 3D bioprinting enables free-form fabrication of complex living tissue structures. In the present work, production of human jawbone models was realised via projection-based stereolithography. Constructs were bioprinted containing primary jawbone-derived osteoblasts and vasculature-like channel structures optionally harbouring primary endothelial cells. After 28 days of cultivation in growth medium or osteogenic medium, expression of cell type-specific markers was confirmed on both the RNA and protein level, while prints maintained their overall structure. Survival of endothelial cells in the printed channels, co-cultured with osteoblasts in medium without supplementation of endothelial growth factors, was demonstrated. Constructs showed not only mineralisation, being one of the characteristics of osteoblasts, but also hinted at differentiation to an osteocyte phenotype. These results indicate the successful biofabrication of an in vitro model of the human jawbone, which presents key features of this special bone entity and hence appears promising for application in jawbone-specific research.TU Berlin, Open-Access-Mittel – 202

Inspired by the human placenta: a novel 3D bioprinted membrane system to create barrier models

Barrier organ models need a scaffold structure to create a two compartment culture. Technical filter membranes used most often as scaffolds may impact cell behaviour and present a barrier themselves, ultimately limiting transferability of test results. In this work we present an alternative for technical filter membrane systems: a 3D bioprinted biological membrane in 24 well format. The biological membrane, based on extracellular matrix (ECM), is highly permeable and presents a natural 3D environment for cell culture. Inspired by the human placenta we established a coculture of a trophoblast-derived cell line (BeWo b30), together with primary placental fibroblasts within the biological membrane (simulating villous stroma) and primary human placental endothelial cells—representing three cellular components of the human placental villus. All cell types maintained their cell type specific marker expression after two weeks of coculture on the biological membrane. In permeability assays the trophoblast layer developed a barrier on the biological membrane, which was even more pronounced when cocultured with fibroblasts. In this work we present a filter membrane free scaffold, we characterize its properties and assess its suitability for cell culture and barrier models. Further we show a novel placenta inspired model in a complex bioprinted coculture. In the absence of an artificial filter membrane, we demonstrate barrier architecture and functionality.TU Berlin, Open-Access-Mittel – 202

Amino Acid and Phospholipid Metabolism as an Indicator of Inflammation and Subtle Cardiomyopathy in Patients with Marfan Syndrome

Patients with Marfan syndrome (MFS) have an increased risk of aortic aneurysm formation, dissection and development of a subtle cardiomyopathy. We analyzed amino acid and lipid metabolic pathways in MFS patients, seeking biomarker patterns as potential monitoring tools of cardiovascular risk with deterioration of myocardial function. We assessed myocardial function in 24 adult MFS patients and compared traditional laboratory values and mass spectrometry-based amino acid, phospholipid and acylcarnitine metabolomes in patients with those in healthy controls. Analytes for which values differed between patients and controls were subjected to regression analysis. A high proportion of patients had signs of impaired diastolic function and elevated serum levels of NT-proBNP. Patients had lower serum levels of taurine, histidine and PCaeC42:3 than controls. The evidence of diastolic dysfunction, aortic root dimensions and history of aortic root surgery correlated with NT-proBNP and taurine levels. Alterations in serum levels of metabolism derived analytes link MFS pathophysiology with inflammation, oxidative stress and incipient cardiomyopathy

Light–Dark Adaptation of Channelrhodopsin Involves Photoconversion between the all-<i>trans</i> and 13-<i>cis</i> Retinal Isomers

Channelrhodopsins (ChR) are light-gated ion channels of green algae that are widely used to probe the function of neuronal cells with light. Most ChRs show a substantial reduction in photocurrents during illumination, a process named “light adaptation”. The main objective of this spectroscopic study was to elucidate the molecular processes associated with light–dark adaptation. Here we show by liquid and solid-state nuclear magnetic resonance spectroscopy that the retinal chromophore of fully dark-adapted ChR is exclusively in an all<i>-trans</i> configuration. Resonance Raman (RR) spectroscopy, however, revealed that already low light intensities establish a photostationary equilibrium between all<i>-trans</i>,15<i>-anti</i> and 13<i>-cis</i>,15-<i>syn</i> configurations at a ratio of 3:1. The underlying photoreactions involve simultaneous isomerization of the C(13)C(14) and C(15)N bonds. Both isomers of this DA_app state may run through photoinduced reaction cycles initiated by photoisomerization of only the C(13)C(14) bond. RR spectroscopic experiments further demonstrated that photoinduced conversion of the apparent dark-adapted (DA_app) state to the photocycle intermediates P500 and P390 is distinctly more efficient for the all-<i>trans</i> isomer than for the 13-<i>cis</i> isomer, possibly because of different chromophore–water interactions. Our data demonstrating two complementary photocycles of the DA_app isomers are fully consistent with the existence of two conducting states that vary in quantitative relation during light–dark adaptation, as suggested previously by electrical measurements

Confirmation of the topology of the Wendelstein 7-X magnetic field to better than 1:100,000

Fusion energy research has in the past 40 years focused primarily on the tokamak concept, but recent advances in plasma theory and computational power have led to renewed interest in stellarators. The largest and most sophisticated stellarator in the world, Wendelstein 7-X (W7-X), has just started operation, with the aim to show that the earlier weaknesses of this concept have been addressed successfully, and that the intrinsic advantages of the concept persist, also at plasma parameters approaching those of a future fusion power plant. Here we show the first physics results, obtained before plasma operation: that the carefully tailored topology of nested magnetic surfaces needed for good confinement is realized, and that the measured deviations are smaller than one part in 100,000. This is a significant step forward in stellarator research, since it shows that the complicated and delicate magnetic topology can be created and verified with the required accuracy