Cryogenic-compatible spherical rotors and stators for magic angle spinning dynamic nuclear polarization

Cryogenic magic angle spinning (MAS) is a standard technique utilized for dynamic nuclear polarization (DNP) in solid-state nuclear magnetic resonance (NMR). Here we describe the optimization and implementation of a stator for cryogenic MAS with 9.5 mm diameter spherical rotors, allowing for DNP experiments on large sample volumes. Designs of the stator and rotor for cryogenic MAS build on recent advancements of MAS spheres and take a step further to incorporate sample insert and eject and a temperature-independent spinning stability of ±1 Hz. At a field of 7 T and spinning at 2.0 kHz with a sample temperature of 105–107 K, DNP enhancements of 256 and 200 were observed for 124 and 223 µL sample volumes, respectively, each consisting of 4 M 13C, 15N-labeled urea and 20 mM AMUPol in a glycerol–water glassy matrix.</p

Cytoskeletal protein kinases: titin and its relations in mechanosensing

Titin, the giant elastic ruler protein of striated muscle sarcomeres, contains a catalytic kinase domain related to a family of intrasterically regulated protein kinases. The most extensively studied member of this branch of the human kinome is the Ca2+–calmodulin (CaM)-regulated myosin light-chain kinases (MLCK). However, not all kinases of the MLCK branch are functional MLCKs, and about half lack a CaM binding site in their C-terminal autoinhibitory tail (AI). A unifying feature is their association with the cytoskeleton, mostly via actin and myosin filaments. Titin kinase, similar to its invertebrate analogue twitchin kinase and likely other “MLCKs”, is not Ca2+–calmodulin-activated. Recently, local protein unfolding of the C-terminal AI has emerged as a common mechanism in the activation of CaM kinases. Single-molecule data suggested that opening of the TK active site could also be achieved by mechanical unfolding of the AI. Mechanical modulation of catalytic activity might thus allow cytoskeletal signalling proteins to act as mechanosensors, creating feedback mechanisms between cytoskeletal tension and tension generation or cellular remodelling. Similar to other MLCK-like kinases like DRAK2 and DAPK1, TK is linked to protein turnover regulation via the autophagy/lysosomal system, suggesting the MLCK-like kinases have common functions beyond contraction regulation

Improving the sensitivity of MAS spheres using a 9.5 mm spherical shell with 219 μL sample volume spinning in a spherical solenoid coil

Spherical rotors in magic angle spinning (MAS) nuclear magnetic resonance (NMR) experiments have potential advantages relative to cylindrical rotors in terms of ease of fabrication, low risk of rotor crash, easy sample exchange, and better microwave access. However, one major disadvantage so far of spherical rotors is poor NMR filling factor due to the small sample volume and large cylindrical radiofrequency (RF) coil. Here we present a novel NMR coil geometry in the form of a spherical coil. The spherical coil best fits the spherical sample to maximize sensitivity, while also providing excellent RF homogeneity. We further improve NMR sensitivity by employing a spherical shell as the rotor, thereby maximizing sample volume (219 μL in this case of 9.5 mm outer diameter spheres). The spinning gas is supplied by a 3D-printed ring stator external to the coil, thereby introducing a simplified form of MAS stators. In this apparatus, the RF field generated along the coil axis is perpendicular to the external magnetic field, regardless of rotor orientation. We observe a linear increase in sensitivity with increasing sample volume. We also simulate the RF performance of spherical and cylindrical solenoid coils with constant or variable pitch for spherical and cylindrical rotors, respectively. The simulation results show that spherical solenoid coils generate comparable B1 field intensities but have better homogeneity than cylindrical solenoid coils do.ISSN:1090-780

Highly stable magic angle spinning spherical rotors

The use of spherical rotors for magic angle spinning offers a number of advantages, including improved sample exchange, efficient microwave coupling for dynamic nuclear polarization nuclear magnetic resonance (NMR) experiments, and, most significantly, high frequency and stable spinning with minimal risk of rotor crash. Here we demonstrate the simple retrofitting of a commercial NMR probe with MAS spheres for solid-state NMR. We analyze a series of turbine groove geometries to investigate the importance of the rotor surface for spinning performance. Of note, rotors lacking any surface modification spin rapidly and stably even without feedback control. The high stability of a spherical rotor about the magic angle is shown to be dependent on its inertia tensor rather than the presence of turbine grooves.</p

PEGylation, increasing specific activity and multiple dosing as strategies to improve the risk-benefit profile of targeted radionuclide therapy with 177Lu-DOTA-bombesin analogues

Background Radiolabelled bombesin (BN) conjugates are promising radiotracers for imaging and therapy of breast and prostate tumours, in which BN2/gastrin-releasing peptide receptors are overexpressed. We describe the influence of the specific activity of a 177Lu-DOTA-PEG5k-Lys-B analogue on its therapeutic efficacy and compare it with its non-PEGylated counterpart. Methods Derivatisation of a stabilised DOTA-BN(7–14)[Cha13,Nle14] analogue with a linear PEG molecule of 5 kDa (PEG5k) was performed by PEGylation of the ϵ-amino group of a β3hLys-βAla-βAla spacer between the BN sequence and the DOTA chelator. The non-PEGylated and the PEGylated analogues were radiolabelled with 177Lu. In vitro evaluation was performed in human prostate carcinoma PC-3 cells, and in vivo studies were carried out in nude mice bearing PC-3 tumour xenografts. Different specific activities of the PEGylated BN analogue and various dose regimens were evaluated concerning their therapeutic efficacy. Results The specificity and the binding affinity of the BN analogue for BN2/GRP receptors were only slightly reduced by PEGylation. In vitro binding kinetics of the PEGylated analogue was slower since steady-state condition was reached after 4 h. PEGylation improved the stability of BN conjugate in vitro in human plasma by a factor of 5.6. The non-PEGylated BN analogue showed favourable pharmacokinetics already, i.e. fast blood clearance and renal excretion, but PEGylation improved the in vivo behaviour further. One hour after injection, the tumour uptake of the PEG5k-BN derivative was higher compared with that of the non-PEGylated analogue (3.43 ± 0.63% vs. 1.88 ± 0.4% ID/g). Moreover, the increased tumour retention resulted in a twofold higher tumour accumulation at 24 h p.i., and increased tumour-to-non-target ratios (tumour-to-kidney, 0.6 vs. 0.4; tumour-to-liver, 8.8 vs. 5.9, 24 h p.i.). In the therapy study, both 177Lu-labelled BN analogues significantly inhibited tumour growth. The therapeutic efficacy was highest for the PEGylated derivative of high specific activity administered in two fractions (2 × 20 MBq = 40 MBq) at day 0 and day 7 (73% tumour growth inhibition, 3 weeks after therapy). Conclusions PEGylation and increasing the specific activity enhance the pharmacokinetic properties of a 177Lu-labelled BN-based radiopharmaceutical and provide a protocol for targeted radionuclide therapy with a beneficial anti-tumour effectiveness and a favourable risk-profile at the same time.ISSN:2191-219

A multi-sample 94GHz dissolution dynamic-nuclear-polarization system

We describe the design and initial performance results of a multi-sample dissolution dynamic-nuclear-polarization (DNP) polarizer based on a Helium-temperature NMR cryostat for use in a wide-bore NMR magnet with a room-temperature bore. The system is designed to accommodate up to six samples in a revolver-style sample changer that allows changing samples at liquid-Helium temperature and at pressures ranging from ambient pressure down to 1mbar. The multi-sample setup is motivated by the desire to do repetitive in vivo measurements and to characterize the DNP process by investigating samples of different chemical composition. The system can be loaded with up to six samples simultaneously to reduce sample loading and unloading. Therefore, series of experiments can be carried out faster and more reliably. The DNP probe contains an oversized microwave cavity and includes EPR and NMR capabilities for monitoring the DNP process. In the solid state, DNP enhancements corresponding to ∼45% polarization for [1-(13)C]pyruvic acid with a trityl radical have been measured. In the initial liquid-state acquisition experiments described here, the polarization was found to be ∼13%, corresponding to an enhancement factor exceeding 16,000 relative to thermal polarization at 9.4T and ambient temperature

Towards optical MAS magnetic resonance using optical traps

Higher magic angle spinning (MAS) frequencies than currently available are desirable to improve spectral resolution in NMR and EPR systems. While conventional strategies employ pneumatic spinning limited by fluid dynamics, this paper demonstrates the development of an optical spinning technique in which vacuum quality dictates the maximum achievable spinning frequency. Using optical traps, we levitated a range of micron-sized samples. Under vacuum we achieved optical rotation of a single ∼10 μm diameter particle of vaterite at several mbar up to hundreds of Hz and of 20 μm diameter SiO2 particles at ≤10−2 mbar at several kHz. At ambient conditions, we optically levitated γ-irradiated alanine particles of 20–50 μm diameter. Additionally, using a single chip EPR detector operating at 11 GHz, we measured the EPR spectrum for a 30 μm γ-irradiated alanine particle in contact with the chip surface (i.e., without optical levitation) in a single scan lasting 92 s. These observations suggest that a γ-irradiated alanine particle having a diameter in the order of 30 μm is a promising candidate for our aim of demonstrating the first magnetic resonance experiment on optically levitated samples. Furthermore, we discuss strategies, limitations, and the potential of implementing MAS with optical traps for NMR and EPR

Biomolecular solid-state NMR spectroscopy at 1200 MHz: the gain in resolution

Progress in NMR in general and in biomolecular applications in particular is driven by increasing magnetic-field strengths leading to improved resolution and sensitivity of the NMR spectra. Recently, persistent superconducting magnets at a magnetic field strength (magnetic induction) of 28.2 T corresponding to 1200 MHz proton resonance frequency became commercially available. We present here a collection of high-field NMR spectra of a variety of proteins, including molecular machines, membrane proteins, viral capsids, fibrils and large molecular assemblies. We show this large panel in order to provide an overview over a range of representative systems under study, rather than a single best performing model system. We discuss both carbon-13 and proton-detected experiments, and show that in C-13 spectra substantially higher numbers of peaks can be resolved compared to 850 MHz while for H-1 spectra the most impressive increase in resolution is observed for aliphatic side-chain resonances.ISSN:0925-2738ISSN:1573-500