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We present a general theoretical description that allows us to describe the influence of isotropic chemical shift in homonuclear and heteronuclear dipolar recoupling experiments in magic-angle-spinning solid-state NMR. Through a transformation of the Hamiltonian into an interaction frame with the combined radio-frequency irradiation and the isotropic chemical shift, we determine an effective Hamiltonian to first order with respect to the relevant internal nuclear spin interactions. This unravels the essential resonance conditions for efficient dipolar recoupling. Furthermore, we propose how to handle situations where the resonance conditions are not exactly fulfilled. To verify the general theoretical description, we compare numerical simulations using a time-sliced time-dependent Hamiltonian with simulations using the calculated effective Hamiltonian for propagation. The comparisons are exemplified for the homonuclear dipolar recoupling experiments C72¹ and POST-C72¹. [ABSTRACT FROM AUTHOR]

We present a theoretical analysis of the influence of chemical shifts on amplitude-modulated heteronuclear dipolar recoupling experiments in solid-state NMR spectroscopy. The method is demonstrated using the Rotor Echo Short Pulse IRrAdiaTION mediated Cross-Polarization (RESPIRATIONCP) experiment as an example. By going into the pulse sequence rf interaction frame and employing a quintuple-mode operator-based Floquet approach, we describe how chemical shift offset and anisotropic chemical shift affect the efficiency of heteronuclear polarization transfer. In this description, it becomes transparent that the main attribute leading to non-ideal performance is a fictitious field along the rf field axis, which is generated from second-order cross terms arising mainly between chemical shift tensors and themselves. This insight is useful for the development of improved recoupling experiments. We discuss the validity of this approach and present quaternion calculations to determine the effective resonance conditions in a combined rf field and chemical shift offset interaction frame transformation. Based on this, we derive a broad-banded version of the RESPIRATIONCP experiment. The new sequence is experimentally verified using SNNFGAILSS amyloid fibrils where simultaneous 15N→ 13CO and 15N→ 13Cα coherence transfer is demonstrated on high-field NMR instrumentation, requiring great offset stability. [ABSTRACT FROM AUTHOR]

A unified strategy of two-pulse based heteronuclear decoupling for solid-state magic-angle spinning nuclear magnetic resonance is presented. The analysis presented here shows that different decoupling sequences like two-pulse phase-modulation (TPPM), X-inverse-X (XiX), and finite pulse refocused continuous wave (rCWA) are basically specific solutions of a more generalized decoupling scheme which incorporates the concept of time-modulation along with phase-modulation. A plethora of other good decoupling conditions apart from the standard, TPPM, XiX, and rCWA decoupling conditions are available from the unified decoupling approach. The importance of combined time- and phasemodulation in order to achieve the best decoupling conditions is delineated. The consequences of different indirect dipolar interactions arising from cross terms comprising of heteronuclear and homonuclear dipolar coupling terms and also those between heteronuclear dipolar coupling and chemicalshift anisotropy terms are presented in order to unfold the effects of anisotropic interactions under different decoupling conditions. Extensive numerical simulation results are corroborated with experiments on standard amino acids. [ABSTRACT FROM AUTHOR]

A novel strategy for heteronuclear dipolar decoupling in magic-angle spinning solid-state nuclear magnetic resonance (NMR) spectroscopy is presented, which eliminates residual static high-order terms in the effective Hamiltonian originating from interactions between oscillating dipolar and anisotropic shielding tensors. The method, called refocused continuous-wave (rCW) decoupling, is systematically established by interleaving continuous wave decoupling with appropriately inserted rotor-synchronized high-power π refocusing pulses of alternating phases. The effect of the refocusing pulses in eliminating residual effects from dipolar coupling in heteronuclear spin systems is rationalized by effective Hamiltonian calculations to third order. In some variants the π pulse refocusing is supplemented by insertion of rotor-synchronized π/2 purging pulses to further reduce the residual dipolar coupling effects. Five different rCW decoupling sequences are presented and their performance is compared to state-of-the-art decoupling methods. The rCW decoupling sequences benefit from extreme broadbandedness, tolerance towards rf inhomogeneity, and improved potential for decoupling at relatively low average rf field strengths. In numerical simulations, the rCW schemes clearly reveal superior characteristics relative to the best decoupling schemes presented so far, which we to some extent also are capable of demonstrating experimentally. A major advantage of the rCW decoupling methods is that they are easy to set up and optimize experimentally. [ABSTRACT FROM AUTHOR]

The past decade has demonstrated increasing interests in using optimal control based methods within coherent quantum controllable systems. The versatility of such methods has been demonstrated with particular elegance within nuclear magnetic resonance (NMR) where natural separation between coherent and dissipative spin dynamics processes has enabled coherent quantum control over long periods of time to shape the experiment to almost ideal adoption to the spin system and external manipulations. This has led to new design principles as well as powerful new experimental methods within magnetic resonance imaging, liquid-state and solid-state NMR spectroscopy. For this development to continue and expand, it is crucially important to constantly improve the underlying numerical algorithms to provide numerical solutions which are optimally compatible with implementation on current instrumentation and at same time are numerically stable and offer fast monotonic convergence toward the target. Addressing such aims, we here present a smoothing monotonically convergent algorithm for pulse sequence design in magnetic resonance which with improved optimization stability lead to smooth pulse sequence easier to implement experimentally and potentially understand within the analytical framework of modern NMR spectroscopy. [ABSTRACT FROM AUTHOR]

Dipolar truncation prevents accurate measurement of long-range internuclear distances between nuclei of the same spin species, e.g., within 13C–13C spin pairs in uniformly 13C-isotope-labeled proteins, using magic-angle spinning solid-state NMR spectroscopy. Accordingly, one of the richest sources of accurate structure information is at present not exploited fully, leaving the bulk part of the experimentally derived structural constraints to less accurate long-range 13C–13C dipolar couplings estimated from methods based on spin diffusion through proton spins in the close environment. In this paper, we extend our previous triple-oscillating field technique [N. Khaneja and N. C. Nielsen, J. Chem. Phys. 128, 015103 (2008)] for dipolar recoupling without dipolar truncation in homonuclear spin systems to a more advanced rf modulation with four independent oscillations and rotations involving nonorthogonal axes. This provides important new degrees of freedom, which are used to improve the scaling factor of the recoupled dipole-dipole couplings by a factor of 2.5 relative to the triple-oscillating field approach. This significant improvement, obtained by refocusing of otherwise defocused parts of the residual dipolar coupling Hamiltonian, may be exploited to measure much weaker 13C–13C dipolar couplings (and thereby longer distances) with much higher accuracy. We present a detailed theoretical description of multiple-field oscillating recoupling experiments, along with numerical simulations and experimental results on U–13C, 15N-L-threonine and U–13C,15N-ubiquitin. [ABSTRACT FROM AUTHOR]

We report the use of optimal control algorithms for tailoring the effective Hamiltonians in nuclear magnetic resonance (NMR) spectroscopy through sophisticated radio-frequency (rf) pulse irradiation. Specifically, we address dipolar recoupling in solid-state NMR of powder samples for which case pulse sequences offering evolution under planar double-quantum and isotropic mixing dipolar coupling Hamiltonians are designed. The pulse sequences are constructed numerically to cope with a range of experimental conditions such as inhomogeneous rf fields, spread of chemical shifts, the intrinsic orientation dependencies of powder samples, and sample spinning. While the vast majority of previous dipolar recoupling sequences are operating through planar double-or zero-quantum effective Hamiltonians, we present here not only improved variants of such experiments but also for the first time homonuclear isotropic mixing sequences which transfers all Ix, Iy, and Iz polarizations from one spin to the same operators on another spin simultaneously and with equal efficiency. This property may be exploited to increase the signal-to-noise ratio of two-dimensional experiments by a factor of √2 compared to conventional solid-state methods otherwise showing the same efficiency. The sequences are tested numerically and experimentally for a powder of 13Cα, 13Cβ-L-alanine and demonstrate substantial sensitivity gains over previous dipolar recoupling experiments. [ABSTRACT FROM AUTHOR]

Oligomeric and protofibrillar aggregates formed by the amyloid-β peptide (Aβ) are believed to be involved in the pathology of Alzheimer's disease. Central to Alzheimer pathology is also the fact that the longer Aβ42 peptide is more prone to aggregation than the more prevalent Aβ40. Detailed structural studies of Aβ oligomers and protofibrils have been impeded by aggregate heterogeneity and instability. We previously engineered a variant of Aβ that forms stable protofibrils and here we use solid-state NMR spectroscopy and molecular modeling to derive a structural model of these. NMR data are consistent with packing of residues 16 to 42 of Aβ protomers into hexameric barrel-like oligomers within the protofibril. The core of the oligomers consists of all residues of the central and C-terminal hydrophobic regions of Aβ, and hairpin loops extend from the core. The model accounts for why Aβ42 forms oligomers and protofibrils more easily than Aβ40. [ABSTRACT FROM AUTHOR]

1H-detected magic-angle spinning NMR experiments facilitate structural biology of solid proteins, which requires using deuterated proteins. However, often amide protons cannot be back-exchanged sufficiently, because of a possible lack of solvent exposure. For such systems, using 2H excitation instead of 1H excitation can be beneficial because of the larger abundance and shorter longitudinal relaxation time, T1, of deuterium. A new structure determination approach, 'quadruple-resonance NMR spectroscopy', is presented which relies on an efficient 2H-excitation and 2H-13C cross-polarization (CP) step, combined with 1H detection. We show that by using 2H-excited experiments better sensitivity is possible on an SH3 sample recrystallized from 30 % H2O. For a membrane protein, the ABC transporter ArtMP in native lipid bilayers, different sets of signals can be observed from different initial polarization pathways, which can be evaluated further to extract structural properties. [ABSTRACT FROM AUTHOR]

Cisplatin [ cis-diamminedichloridoplatinum(II)] is used in chemotherapy where platinum–DNA adducts initiate tumor cell death. It is possible that side effects such as neurotoxicity and cellular cisplatin resistance can be due to interaction of cisplatin with lipids and the phospholipid bilayer. In this study, 13C, 31P, and 15N solid-state NMR spectra of 1-palmitoyl-2-oleoyl phosphatidylserine (POPS) bilayers, POPS bilayers with 10 mol% cisplatin, and POPS bilayers with 30 mol% cisplatin were acquired. In addition, 15N{31P} rotational echo double resonance spectra of POPS bilayers with 30 mol% cisplatin were acquired. The data demonstrate that the serine head group of phosphatidylserine binds to the aquated form of cisplatin and that cisplatin release of ammine takes place. It appears that the cisplatin release of ammine is followed by another cisplatin–POPS complex formation, possibly with cisplatin binding to one of the oxygen atoms of the POPS phosphate moiety. [ABSTRACT FROM AUTHOR]

Background: Creatine is a key intermediate in energy metabolism and supplementation of creatine has been used for increasing muscle mass, strength and endurance. Creatine supplementation has also been reported to trigger the skeletal muscle expression of insulin like growth factor I, to increase the fat-free mass and improve cognition in elderly, and more explorative approaches like transcriptomics has revealed additional information. The aim of the present study was to reveal additional insight into the biochemical effects of creatine supplementation at the protein and metabolite level by integrating the explorative techniques, proteomics and NMR metabonomics, in a systems biology approach. Methods: Differentiated mouse myotube cultures (C2C12) were exposed to 5 mM creatine monohydrate (CMH) for 24 hours. For proteomics studies, lysed myotubes were analyzed in single 2-DGE gels where the first dimension of protein separation was pI 5-8 and second dimension was a 12.5% Criterion gel. Differentially expressed protein spots of significance were excised from the gel, desalted and identified by peptide mass fingerprinting using MALDI-TOF MS. For NMR metabonomic studies, chloroform/methanol extractions of the myotubes were subjected to one-dimensional 1H NMR spectroscopy and the intracellular oxidative status of myotubes was assessed by intracellular DCFH2 oxidation after 24 h pre-incubation with CMH. Results: The identified differentially expressed proteins included vimentin, malate dehydrogenase, peroxiredoxin, thioredoxin dependent peroxide reductase, and 75 kDa and 78 kDa glucose regulated protein precursors. After CMH exposure, up-regulated proteomic spots correlated positively with the NMR signals from creatine, while downregulated proteomic spots were negatively correlated with these NMR signals. The identified differentially regulated proteins were related to energy metabolism, glucose regulated stress, cellular structure and the antioxidative defence system. The suggested improvement of the antioxidative defence was confirmed by a reduced intracellular DCFH2 oxidation with increasing concentrations of CMH in the 24 h pre-incubation medium. Conclusions: The explorative approach of this study combined with the determination of a decreased intracellular DCFH2 oxidation revealed an additional stimulation of cellular antioxidative mechanisms when myotubes were exposed to CMH. This may contribute to an increased exercise performance mediated by increased ability to cope with training-induced increases in oxidative stress. [ABSTRACT FROM AUTHOR]

Abstract: The structure of the chlorosome baseplate protein CsmA from Chlorobium tepidum in a 1:1 chloroform:methanol solution was determined using liquid-state NMR spectroscopy. The data reveal that the 59-residue protein is predominantly α-helical with a long helical domain extending from residues V6 to L36, containing a putative bacteriochlorophyll a binding domain, and a short helix in the C-terminal part extending from residues M41 to G49. These elements are compatible with a model of CsmA having the long N-terminal α-helical stretch immersed into the lipid monolayer confining the chlorosome and the short C-terminal helix protruding outwards, thus available for interaction with the Fenna–Matthews–Olson antenna protein. [Copyright &y& Elsevier]

Frequent exposure of terrestrial insects to temperature variation has led to the evolution of protective biochemical and physiological mechanisms, such as the heat shock response, which markedly increases the tolerance to heat stress. Insight into such mechanisms has, so far, mainly relied on selective studies of specific compounds or characteristics or studies at the genomic or proteomic levels. In the present study, we have used untargeted NMR metabolomic profiling to examine the biological response to heat stress in Drosophila melanogaster. The metabolite profile was analyzed during recovery after exposure to different thermal stress treatments and compared with untreated controls. Both moderate and severe heat stress gave clear effects on the metabolite profiles. The profiles clearly demonstrated that hardening by moderate heat stress led to a faster reestablishment of metabolite homeostasis after subsequent heat stress. Several metabolites were identified as responsive to heat stress and could be related to known physiological and biochemical responses. The time course of the recovery of metabolite homeostasis mirrored general changes in gene expression, showing that recovery follows the same temporal pattern at these two biological levels. Finally, our data show that heat hardening permits a quicker return to homeostasis, rather than a reduction of the acute metabolic perturbation and that the reestablishment of homeostasis is important for obtaining maximal heat-hardening effect. The results display the power of NMR metabolomic profiling for characterization of the instantaneous physiological condition, enabling direct visualization of the perturbation of and return to homeostasis. [ABSTRACT FROM AUTHOR]

We present a bimodal Floquet analysis of the recently introduced refocused continuous wave ( r CW) solid-state NMR heteronuclear dipolar decoupling method and compare it with the similar looking X-inverse X (XiX) scheme. The description is formulated in the rf interaction frame and is valid for both finite and ideal π pulse r CW irradiation that forms the refocusing element in the r CW scheme. The effective heteronuclear dipolar coupling Hamiltonian up to first order is described. The analysis delineates the difference between the two sequences to different orders of their Hamiltonians for both diagonal and off-diagonal parts. All the resonance conditions observed in experiments and simulations have been characterised and their influence on residual line broadening is highlighted. The theoretical comparison substantiates the numerical simulations and experimental results to a large extent. [ABSTRACT FROM AUTHOR]

Abstract: Refocused continuous wave (rCW) decoupling is presented as an efficient and robust means to obtain well-resolved magic-angle-spinning solid-state NMR spectra of low-γ spins, such as 13C dipolar coupled to fluorine. The rCW decoupling sequences, recently introduced for 1H decoupling, are very robust towards large isotropic and anisotropic shift ranges as often encountered for 19F spins. In rCW decoupling, the so-called refocusing pulses inserted into the CW irradiation eliminate critical residual second- and third-order dipolar coupling and dipolar-coupling against chemical shielding anisotropy cross-terms in the effective Hamiltonian through time-reversal (i.e. refocusing). As important additional assets, the rCW decoupling sequences are robust towards variations in rf amplitudes, operational at low to high spinning speeds, and easy to set-up for optimal performance experimentally. These aspects are demonstrated analytically/numerically and experimentally in comparison to state-of-the-art decoupling sequences such as TPPM, SPINAL-64, and frequency-swept variants of these. [Copyright &y& Elsevier]

Abstract: Three different techniques (adiabatic passage Hartman-Hahn cross-polarization, optimal control designed pulses, and EXPORT) are compared for transferring 15N magnetization to 13C 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-13C,15N labeled samples and are generally superior for samples with non-uniform labeling schemes (such as 1,3- and 2-13C 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. [Copyright &y& Elsevier]

Abstract: A computer program for fast and accurate numerical simulation of solid-state NMR experiments is described. The program is designed to emulate a NMR spectrometer by letting the user specify high-level NMR concepts such as spin systems, nuclear spin interactions, RF irradiation, free precession, phase cycling, coherence-order filtering, and implicit/explicit acquisition. These elements are implemented using the Tel scripting language to ensure a minimum of programming overhead and direct interpretation without the need for compilation, while maintaining the flexibility of a full-featured programming language. Basicly, there are no intrinsic limitations to the number of spins, types of interactions, sample conditions (static or spinning, powders, uniaxially oriented molecules, single crystals, or solutions), and the complexity or number of spectral dimensions for the pulse sequence. The applicability ranges from simple ID experiments to advanced multiple-pulse and multiple-dimensional experiments, series of simulations, parameter scans, complex data manipulation/visualization, and iterative fitting of simulated to experimental spectra. A major effort has been devoted to optimizing the computation speed using state-of-the-art algorithms for the time-consuming parts of the calculations implemented in the core of the program using the C programming language. Modification and maintenance of the program are facilitated by releasing the program as open source software (General Public License) currently at http://nmr.imsb.au.dk. The general features of the program are demonstrated by numerical simulations of various aspects for REDOR, rotational resonance, DRAMA, DRAWS, HORROR, C7, TEDOR, POST-C7, CW decoupling, TPPM, F-SLG, SLF, SEMA-CP, PISEMA, RFDR, QCPMG-MAS, and MQ-MAS experiments. [Copyright &y& Elsevier]

Abstract: Aimed at performing dipolar recoupling as part of biological solid-state NMR experiments without using excessively strong rf irradiation, we present a series of low-power supercycled symmetry-based and exponentially modulated pulse sequences facilitating efficient recoupling over specified chemical shift ranges even at high-speed spinning conditions. The experiments are designed through simple phase modulation of known ‘lower-quality’ symmetry-based pulse sequences or highly rf-demanding exponentially modulated recoupling experiments. The methods are described analytically and numerically and demonstrated experimentally by one- and two-dimensional correlation spectra of 2,3-13C2-L-alanine and the fibrillating-core decapeptide SNNFGAILSS of human Islet Amyloid Poly-Peptide (hIAPP, or amylin) uniformly 13C,15N-labeled at the FGAIL residues. [Copyright &y& Elsevier]

Cation binding to Na,K-ATPase is characterized in native membranes at room temperature by solid-state NMR spectroscopy using the K+ congener 205Tl. It has been demonstrated that the signals from occluded Tl+ and nonspecifically bound Tl+ can be detected and distinguished by NMR. Effects of dipole-dipole coupling between ¹H and 205Tl in the occlusion sites show that the ions are rigidly bound, rather than just occluded. Furthermore, a low chemical shift suggests occlusion site geometries with a relatively small contribution from carboxylate and hydroxyl groups. Nonspecific binding of Tl+ is characterized by rapid chemical exchange, in agreement with the observed low binding affinity. [ABSTRACT FROM AUTHOR]

The cellular functions of several S100 proteins involve specific interactions with phospholipids and the cell membrane. The interactions between calbindin D9k (S100D) and the detergent dodecyl phosphocholine (DPC) were studied using NMR spectroscopy. In the absence of Ca2+, the protein associates with DPC micelles. The micelle-associated state has intact helical secondary structures but no apparent tertiary fold. At neutral pH, Ca2+-loaded calbindin D9k does not associate with DPC micelles. However, a specific interaction is observed with individual DPC molecules at a site close to the linker between the two EF-hands. Binding to this site occurs only when Ca2+ is bound to the protein. A reduction in pH in the absence of Ca2+ increases the stability of the micelle-associated state. This along with the corresponding reduction in Ca2+ affinity causes a transition to the micelle-associated state also in the presence of Ca2+ when the pH is lowered. Site-specific analysis of the data indicates that calbindin D9k has a core of three tightly packed helices (A, B, and D), with a dynamic fourth helix (C) more loosely associated. Evidence is presented that the Ca2+-binding characteristics of the two EF-hands are distinctly different in a micelle environment. The role of calbindin D9k in the cell is discussed, along with the broader implications for the function of the S100 protein family. [ABSTRACT FROM AUTHOR]

Abstract: We present a historical recollection on the development of the software package SIMPSON (SIMulation Package for SOlid-state Nmr). This covers a brief description of the underlying ideas and events leading to creation of SIMPSON and numerous auxiliary programs as well as comments on its impact on the development and application of solid-state NMR in research laboratories world-wide. [Copyright &y& Elsevier]

The successful application of solid-state NMR spectroscopy for structural study of biological macromolecules requires high spectral resolution. In presence of abundant H 1 spins, the resolution of the prevailing C 13 or N 15 chemical shift encoding experiments critically depends on the availability of efficient and robust heteronuclear decoupling methods in addition to the use of high-field instrumentation and fast sample spinning. Robustness of the decoupling method towards alterations in amplitude/offset of radio frequency fields due to varying sample states is important to ensure recording of spectra with high resolution over long sampling periods for insensitive samples. Here, we present a phase-alternated refocused continuous-wave decoupling method offering better resolution, easier setup, and higher robustness than previous methods. Improved decoupling is in part ascribed to more efficient cancellation of the residual heteronuclear, H 1 – C 13 , dipolar coupling interactions which are induced by homonuclear, H 1 – H 1 , dipolar coupling interactions. [ABSTRACT FROM AUTHOR]

We present new non-rotor-synchronized variants of the recently introduced refocused continuous wave ( r CW) heteronuclear decoupling method significantly improving the performance relative to the original rotor-synchronized variants. Under non-rotor-synchronized conditions the r CW decoupling sequences provide more efficient decoupling, are easier to setup, and prove more robust towards experimental parameters such as radio frequency (rf) field amplitude and spinning frequency. This is demonstrated through numerical simulations substantiated with experimental results under different sample spinning and rf field amplitude conditions for powder samples of U- 13 C-glycine and U- 13 C- L -histidine·HCl·H 2 O. [ABSTRACT FROM AUTHOR]

Conducting large-scale solid-state NMR simulations requires fast computer software potentially in combination with efficient computational resources to complete within a reasonable time frame. Such simulations may involve large spin systems, multiple-parameter fitting of experimental spectra, or multiple-pulse experiment design using parameter scan, non-linear optimization, or optimal control procedures. To efficiently accommodate such simulations, we here present an improved version of the widely distributed open-source SIMPSON NMR simulation software package adapted to contemporary high performance hardware setups. The software is optimized for fast performance on standard stand-alone computers, multi-core processors, and large clusters of identical nodes. We describe the novel features for fast computation including internal matrix manipulations, propagator setups and acquisition strategies. For efficient calculation of powder averages, we implemented interpolation method of Alderman, Solum, and Grant, as well as recently introduced fast Wigner transform interpolation technique. The potential of the optimal control toolbox is greatly enhanced by higher precision gradients in combination with the efficient optimization algorithm known as limited memory Broyden–Fletcher–Goldfarb–Shanno. In addition, advanced parallelization can be used in all types of calculations, providing significant time reductions. SIMPSON is thus reflecting current knowledge in the field of numerical simulations of solid-state NMR experiments. The efficiency and novel features are demonstrated on the representative simulations. [ABSTRACT FROM AUTHOR]

Highlights: [•] We developed a method of high yield production of recombinant human amylin. [•] Fibril growth and cytotoxicity of recombinant amylin was demonstrated. [•] Recombinant amylin is a well suited material for structural investigations by NMR. [Copyright &y& Elsevier]

Abstract: Polarization transfer between spin-1/2 nuclei and quadrupolar spin-1 nuclei such as N in solid-state NMR is severely challenged by the typical presence of large quadrupole coupling interactions. This has effectively prevented the use of the abundant N spin as a probe to structural information and its use as an element in multi-dimensional solid-state NMR correlation experiments for assignment and structural characterization. In turn, this has been a contributing factor to the extensive use of isotope labeling in biological solid-state NMR, where N is replaced with N. The alternative strategy of using the abundant N spins calls for methods enabling efficient polarization transfer between N and its binding partners. This work demonstrates that the recently introduced CP transfer method can be optimized to achieve efficient H N polarization transfer under magic angle spinning conditions. The method is demonstrated numerically and experimentally on powder samples of NH4NO3 and l-alanine. [Copyright &y& Elsevier]

Wepresent a method to calculate 31P solid-state NMRspectra based on the dynamic input from extended molecular dynamics(MD) simulations. The dynamic information confered by MD simulationsis much more comprehensive than the information provided by traditionalNMR dynamics models based on, for example, order parameters. Therefore,valuable insight into the dynamics of biomolecules may be achievedby the present method. We have applied this method to study the dynamicsof lipid bilayers containing the antimicrobial peptide alamethicin,and we show that the calculated 31P spectra obtained withinput from MD simulations are in agreement with experiments undera large range of different sample conditions, including vesicles andoriented samples with and without peptides. We find that the changesin the 31P spectra upon addition of peptide stem from lipidswith reduced diffusion due to peptide–lipid interactions. [ABSTRACT FROM AUTHOR]

Highlights: [•] Low-field MAS solid-state NMR at 0.45T. [•] Reduced anisotropic shielding effects at low field. [•] Increased second-order quadrupolar couplings at low-field. [•] 23Na second-order quadrupolar lineshape of NaNO3. [•] 31P–19F dipole–dipole coupling of K2PO3F. [ABSTRACT FROM AUTHOR]

Rapid developments in solid-state NMR methodologyhave boosted this technique into a highly versatile tool for structural biology. The invention of increasingly advanced rf pulse sequences that take advantage of better hardware and sample preparation have played an important part in these advances. In the development of these new pulse sequences, researchers have taken advantage of analytical tools, such as average Hamiltonian theory or lately numerical methods based on optimal control theory. [ABSTRACT FROM AUTHOR]

Abstract: Aimed at 13C metabolic magnetic resonance imaging (MRI) and spectroscopy (MRS) applications, we demonstrate that dynamic nuclear polarization (DNP) may be combined with optimal control 2D spatial selection to simultaneously obtain high sensitivity and well-defined spatial restriction. This is achieved through the development of spatial-selective single-shot spiral-readout MRI and MRS experiments combined with dynamic nuclear polarization hyperpolarized [1-13C]pyruvate on a 4.7T pre-clinical MR scanner. The method stands out from related techniques by facilitating anatomic shaped region-of-interest (ROI) single metabolite signals available for higher image resolution or single-peak spectra. The 2D spatial-selective rf pulses were designed using a novel Krotov-based optimal control approach capable of iteratively fast providing successful pulse sequences in the absence of qualified initial guesses. The technique may be important for early detection of abnormal metabolism, monitoring disease progression, and drug research. [Copyright &y& Elsevier]

Abstract: Cold tolerance and metabolic responses to freezing of three slug species common in Scandinavia (Arion ater, Arion rufus and Arion lusitanicus) are reported. Autumn collected slugs were cold acclimated in the laboratory and subjected to freezing conditions simulating likely winter temperatures in their habitat. Slugs spontaneously froze at about −4°C when cooled under dry conditions, but freezing of body fluids was readily induced at −1°C when in contact with external ice crystals. All three species survived freezing for 2days at −1°C, and some A. rufus and A. lusitanicus also survived freezing at −2°C. 1H NMR spectroscopy revealed that freezing of body fluids resulted in accumulation of lactate, succinate and glucose. Accumulation of lactate and succinate indicates that ATP production occurred via fermentative pathways, which is likely a result of oxygen depletion in frozen tissues. Glucose increased from about 6 to 22μg/mg dry tissue upon freezing in A. rufus, but less so in A. ater and A. lusitanicus. Glucose may thus act as a cryoprotectant in these slugs, although the concentrations are not as high as reported for other freeze tolerant invertebrates. [Copyright &y& Elsevier]

Abstract: Heteronuclear dipolar recoupling experiments without 1H decoupling based on simple cross polarization are introduced for applications in biological solid-state NMR. It is shown that standard or adiabatic variants of the cross-polarization experiment with irradiation on the low-γ (e.g., 13C,15N) spins even at modest spinning frequencies enable efficient band-selective or broadband dipolar recoupling without the need for intense 1H decoupling. This facilitates experiments on expensive isotope-labelled protein samples for which sample heating by intense 1H decoupling may lead to sample detoriation. The principle is demonstrated numerically and experimentally on uniformly 13C,15N-labelled samples of GB1 and fibrils of hIAPP from the human islet amyloid labelled on the FGAIL part. [Copyright &y& Elsevier]

Abstract: We present the implementation of optimal control into the open source simulation package SIMPSON for development and optimization of nuclear magnetic resonance experiments for a wide range of applications, including liquid- and solid-state NMR, magnetic resonance imaging, quantum computation, and combinations between NMR and other spectroscopies. Optimal control enables efficient optimization of NMR experiments in terms of amplitudes, phases, offsets etc. for hundreds-to-thousands of pulses to fully exploit the experimentally available high degree of freedom in pulse sequences to combat variations/limitations in experimental or spin system parameters or design experiments with specific properties typically not covered as easily by standard design procedures. This facilitates straightforward optimization of experiments under consideration of rf and static field inhomogeneities, limitations in available or desired rf field strengths (e.g., for reduction of sample heating), spread in resonance offsets or coupling parameters, variations in spin systems etc. to meet the actual experimental conditions as close as possible. The paper provides a brief account on the relevant theory and in particular the computational interface relevant for optimization of state-to-state transfer (on the density operator level) and the effective Hamiltonian on the level of propagators along with several representative examples within liquid- and solid-state NMR spectroscopy. [Copyright &y& Elsevier]

Abstract: The freeze tolerance and accumulation of cryoprotectants was investigated in three geographically different populations of the enchytraeid Enchytraeus albidus (Oligochaeta). E. albidus is widely distributed from the high Arctic to temperate Western Europe. Our results show that E. albidus is freeze tolerant, with freeze tolerance varying extensively between Greenlandic and European populations. Two populations from sub Arctic (Nuuk) and high Arctic Greenland (Zackenberg) survived freezing at −15°C, whereas only 30% of a German population survived this temperature. When frozen, E. albidus responded by catabolising glycogen to glucose, which likely acted as a cryoprotectant. The average glucose concentrations were similar in the three populations when worms were frozen at −2°C, approximately 50μg glucose mg−1 tissue dry weight (DW). At −14°C the glucose concentrations increased to between 110 and 170μgmg−1 DW in worms from Greenland. The average glycogen content of worms from Zackenberg and Nuuk were about 300μgmg−1 DW, but only 230μgmg−1 DW in worms from Germany showing that not all glycogen was catabolised during the experiment. Nuclear magnetic resonance spectrometry (NMR) was used to screen for other putative cryoprotectants. Proline, glutamine and alanine were up regulated in frozen worms at −2°C but only in relatively small concentrations suggesting that they were of little significance for freeze survival. The present study confirms earlier reports that freeze tolerant enchytraeids, like other freeze tolerant oligochaete earthworms, accumulate high concentrations of glucose as a primary cryoprotectant. [Copyright &y& Elsevier]

While the population genetics of inbreeding is fairly well understood, the effects of inbreeding on the physiological and biochemical levels are not. Here we have investigated the effects of inbreeding on the Drosophila melanogaster metabolome. Metabolite fingerprints in males from five outbred and five inbred lines were studied by nuclear magnetic resonance spectroscopy after exposure to benign temperature, heat stress, or cold stress. In both the absence and the presence of temperature stress, metabolite levels were significantly different among inbred and outbred lines. The major effect of inbreeding was increased levels of maltose and decreased levels of 3-hydroxykynurenine and a galactoside [1-O-(4-O-(2-aminoethyl phosphate)-β-D-galactopyranosyl)-x-glycerol] synthesized exclusively in the paragonial glands of Drosophila species, including D. melanogaster. The metabolomic effect of inbreeding at the benign temperature was related to gene expression data from the same inbred and outbred lines. Both gene expression and metabolite data indicate that fundamental metabolic processes are changed or modified by inbreeding. Apart from affecting mean metabolite levels, inbreeding led to an increased between-line variation in metabolite profiles compared to outbred lines. In contrast to previous observations revealing interactions between inbreeding and environmental stress on gene expression patterns and life-history traits, the effect of inbreeding on the metabolite profile was similar across the different temperature treatments. [ABSTRACT FROM AUTHOR]

Abstract: A short exposure to a mild cold stress is sufficient to increase cold tolerance in many insects. This phenomenon, termed rapid cold hardening (RCH) expands the thermal interval that can be exploited by the insect. To investigate the possible role of altered metabolite levels during RCH, the present study used untargeted 1H NMR metabolomic profiling to examine the metabolomic response in Drosophila melanogaster during the 72h following RCH and cold shock treatment. These findings are discussed in relation to the costs and benefits of RCH that are measured in terms of survival and reproductive output. Cold shock caused a persistent disturbance of the metabolite profile that correlated well with a delayed onset of cold shock mortality. The disruption of metabolite homeostasis was smaller following RCH, where control levels were fully recovered after 72h. RCH improved both survival and reproductive output after a subsequent cold shock but the RCH treatment alone was associated with costs in terms of reduced survival and reproductive output. The most pronounced changes following the RCH treatment were elevated levels of glucose and trehalose. Although, it is difficult to discern if a change in a specific metabolite is linked to physiological processes of adaptive, neutral or detrimental nature we observed that the onset and magnitude of the increased sugar levels correlated tightly with the improved chill tolerance following RCH. These findings suggest a putative role of cryoprotectants during RCH which are discussed in the light of the existing literature on the mechanistic background of RCH. [Copyright &y& Elsevier]

We present the use of 2H magic-angle spinning (MAS) NMR on methyl-deuterated α-amino isobutyric acid (Aib) as a new method to obtain fast and accurate structural constraints on peptaibols in membrane-bound environments. Using nonoriented vesicle-reconstituted samples we avoid the delicate preparation of oriented samples, and the use of MAS ensures high sensitivity and thereby very fast acquisition of experimental spectra. Furthermore, given the high content (~40%) of Aib in peptaibols and the fact that the amino acid Aib may be synthesized from cheap starting materials, even in the case of 2H, or 13C, or 15N labeling, this method is ideally suited for studies of the membrane-bound conformation of peptaibols. [ABSTRACT FROM AUTHOR]

Abstract: We present novel pulse sequences for magic-angle-spinning solid-state NMR structural studies of 13C,15N-isotope labeled proteins. The pulse sequences have been designed numerically using optimal control procedures and demonstrate superior performance relative to previous methods with respect to sensitivity, robustness to instrumental errors, and band-selective excitation profiles for typical biological solid-state NMR applications. Our study addresses specifically 15N to 13C coherence transfers being important elements in spectral assignment protocols for solid-state NMR structural characterization of uniformly 13C,15N-labeled proteins. The pulse sequences are analyzed in detail and their robustness towards spin system and external experimental parameters are illustrated numerically for typical 15N–13C spin systems under high-field solid-state NMR conditions. Experimentally the methods are demonstrated by 1D 15N→ 13C coherence transfer experiments, as well as 2D and 3D 15N,13C and 15N,13C,13C chemical shift correlation experiments on uniformly 13C,15N-labeled ubiquitin. [Copyright &y& Elsevier]

Metabonomic analysis of urine utilizing high-resolution NMR spectroscopy and chemometric techniques has proven valuable in characterizing the biochemical response to an intervention. To assess the effect of magnetic field strength on information contained in NMR-based metabonomic data sets, ¹H NMR spectra were acquired on 250-, 400-, 500-, and 800-MHz instruments, respectively, on the same set of human urine samples collected before and after dietary interventions with milk and with meat proteins. Partial least-squares regression discriminant analyses (PLS-DA) were performed in order to elucidate the ability of the ¹H spectra acquired at various field strengths to identity possible spectral differences and discriminate between pre- and postintervention samples. The loadings from PLS-DA contained the same spectral regions, implying that the same metabolites were involved in the discrimination independent of magnetic field strength. The investigation revealed a strong increase in prediction performance and thereby spectral information content when increasing the magnetic field strength from 250 to 500 MHz, while from 500 to 800 MHz the increase was less pronounced. [ABSTRACT FROM AUTHOR]

Abstract: Novel low-power pulse sequences for homonuclear dipolar recoupling in biological solid-state NMR spectroscopy are presented. The pulse sequences are developed numerically using the Simpson simulation program in combination with optimal control procedures. The quality criteria for the experiment optimizations are the achievement of the highest possible sensitivity for powder samples under typical experimental inhomogeneous radio-frequency (rf) field conditions, while keeping the rf field strength sufficiently low to prevent undesirable sample heating in biological applications. The new dipolar recoupling methods are demonstrated numerically and experimentally by double-quantum filtration magic-angle spinning NMR experiments for which sensitivity gains approaching a factor of 2 is observed. [Copyright &y& Elsevier]

Abstract: We report for the first time, oriented-sample solid-state NMR experiments, specifically polarization inversion spin exchange at the magic angle (PISEMA) and 1H–15N heteronuclear chemical shift correlation (HETCOR), applied to an integral seven-transmembrane protein, bacteriorhodopsin (bR), in natural membranes. The spectra of [15N]Met-bR revealed clearly distinguishable signals from the helical and loop regions. By deconvolution of the helix resonances, it was possible to establish constraints for some helix tilt angles. It was estimated that the extracellular section of helix B has a tilt of less than 5° from the membrane normal, while the tilt of helix A was estimated to be 18–22°, both of which are in agreement with most crystal structures. Comparison of the experimental PISEMA spectrum with simulated spectra based on crystal structures showed that PISEMA and HETCOR experiments are extremely sensitive to the polytopic protein structure, and the solid-state NMR spectra for membrane-embedded bR matched most favorably with the recent 1FBB electron crystallography structure. These results suggest that this approach has the potential to yield structural and orientational constraints for large integral polytopic proteins whilst intercalated and functionally competent in a natural membrane. [Copyright &y& Elsevier]

The past few years have witnessed considerable progress in solid-state NMR toward atomic-resolution structural analysis of biological macromolecules. This is relevant for membrane proteins, protein assemblies, and enzyme--substrate complexes not earlier amenable for structural analysis because of low molecular tumbling or lack of suitable crystals. The success of biological solid-state NMR critically depends on the performance of the recoupling methods used as building blocks in the multiple-dimensional experiments. They have to provide the most efficient transfer within the shortest possible time while being sufficiently specific that loss of coherence by transfer to undesired spins is avoided.