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Abstract: Efficient sampling of signals is a key issue for multiple-dimensional NMR experiments to establish the best ratio between experiment time and spectral quality. Focussing on the most widely used sampling strategy using standard rectangular sampling and data analysis by Fourier transformation, a central question is concerned with determining the optimal maximum sampling time in the individual dimensions. The spectral resolution depends directly on this choice, as do the overall experiment times when addressing the indirect dimensions. We present a theoretical, numerical, and experimental analysis of the sampling space problem and propose approaches to efficient sampling for typical cases. [Copyright &y& Elsevier]

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]

The paper describes a family of novel recoupling pulse sequences, called three pulse recoupling. These pulse sequences can be employed for both homonuclear and heteronuclear recoupling experiments and are robust to dispersion in chemical shifts and rf-inhomogeneity. These recoupling pulse sequences can be used in design of two-dimensional solid state NMR experiments that use powdered dephased antiphase coherence ( γ preparation) to encode chemical shifts in the indirect dimension. Both components of this chemical shift encoded gamma-prepared states can be refocused into inphase coherence by a recoupling element. This helps to achieve sensitivity enhancement in 2D NMR experiments by quadrature detection. [ABSTRACT FROM AUTHOR]

Low-field NMR sensor technology is proposed for accurate and operationally simple on-farm or laboratory determination of protein and phosphorus constituents in livestock feed. The total phosphorus content is determined directly on the native sample, while quantification of digestible protein involves enzymatic digestion here adapted to provide the total protein content. Comparison with traditional laboratory reference analysis for the total protein and phosphorus contents of 31 feed samples (including various grains, feed mixtures, and commercial feed products for cattle, pigs, horses, poultry, and sheep) demonstrate the feasibility and good accuracy of the NMR measurements. Table of contents entry – for TOC only. [Display omitted] • Mobile, multinuclear, low-field NMR for on-farm measurements. • Total/digestible protein and total phosphorus contents. • Direct analysis of bulk sample (raw material for phosphorus, digested for protein). • Feasibility and accuracy demonstrated on feed for a wide range of animals. [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: A new experiment, clean HMBC, is introduced for suppression of strong-coupling induced artifacts in HMBC spectra. The culprits of these artifacts are an inherent shortcoming of low-pass J filters in the presence of strong coupling and the 1H π pulse in the middle of the evolution period aimed at suppressing evolution under heteronuclear J couplings and 1H chemical shifts. A π pulse causes coherence transfer in strongly coupled spin systems and, as is well known in e.g., homonuclear J spectra, this leads to peaks that would not be there in the absence of strong coupling. Similar artifacts occur in HMBC spectra, but they have apparently been overlooked, presumably because they have been assigned to inefficiency of low-pass J filters or not noticed because of a coarse digital resolution in the spectra. Clean HMBC is the HMBC technique of choice for molecules notorious for strong coupling among protons, such as carbohydrates, and the new technique is demonstrated on D-mannose. Finally, a fundamental difference between HMBC and H2BC explains why strong-coupling artifacts are much less of a problem in the latter type of spectra. [Copyright &y& Elsevier]

Abstract: A novel procedure for reconstruction of 2D separated-local-field (SLF) NMR spectra from projections of 1D NMR data is presented. The technique, dubbed SLF projection reconstruction from one-dimensional spectra (SLF-PRODI), is particularly useful for uniaxially oriented membrane protein samples and represents a fast and robust alternative to the popular PISEMA experiment which correlates 1H–15N dipole–dipole couplings with 15N chemical shifts. The different 1D projections in the SLF-PRODI experiment are obtained from 1D spectra recorded under influence of homonuclear decoupling sequences with different scaling factors for the heteronuclear dipolar couplings. We demonstrate experimentally and numerically that as few as 2–4 1D projections will normally be sufficient to reconstruct a 2D SLF-PRODI spectrum with a quality resembling typical PISEMA spectra, leading to significant reduction of the acquisition time. [Copyright &y& Elsevier]

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]

There is an increasing need for development of advanced radio-frequency (RF) pulse techniques in modern magnetic resonance imaging (MRI) systems driven by recent advancements in ultra-high magnetic field systems, new parallel transmit/receive coil designs, and accessible powerful computational facilities. 2D spatially selective RF pulses are an example of advanced pulses that have many applications of clinical relevance, e.g., reduced field of view imaging, and MR spectroscopy. The 2D spatially selective RF pulses are mostly generated and optimised with numerical methods that can handle vast controls and multiple constraints. With this study we aim at demonstrating that numerical, optimal control (OC) algorithms are efficient for the design of 2D spatially selective MRI experiments, when robustness towards e.g. field inhomogeneity is in focus. We have chosen three popular OC algorithms; two which are gradient-based, concurrent methods using first- and second-order derivatives, respectively; and a third that belongs to the sequential, monotonically convergent family. We used two experimental models: a water phantom, and an in vivo human head. Taking into consideration the challenging experimental setup, our analysis suggests the use of the sequential, monotonic approach and the second-order gradient-based approach as computational speed, experimental robustness, and image quality is key. All algorithms used in this work were implemented in the MATLAB environment and are freely available to the MRI community. [ABSTRACT FROM AUTHOR]

Radio frequency (RF) spectrally selective multiband pulses or tagging pulses, are applicable in a broad range of magnetic resonance methods. We demonstrate through simulations and experiments a new phase-modulation-only RF pulse for RF tagging based on the Frank poly-phase perfect sequence. In addition, we introduce an extended version with a WURST modulation (Frank-WURST). The new pulses exhibit interesting and flexible spin tagging properties and are easily implemented in existing MR sequences, where they can substitute slice-selective pulses with no additional alterations. [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]

Highlights: [•] A modification of the basic Two-Pulse Phase-Modulation (TPPM) heteronuclear decoupling is proposed. [•] The design of the new scheme, rTPPM, is similar to that of the recently introduced rCW schemes. [•] The decoupling efficiency of the new sequence is tested with simulations and corroborated with experimental findings. [•] The robustness of the newly introduced scheme is compared to that of a few related decoupling methods. [ABSTRACT FROM AUTHOR]

Highlights: [•] Synthesis of a potential angiogenesis-targeted nanoparticle. [•] Assessment of physicochemical and biological properties. [•] Non-invasive evaluation of accumulation in a novel murine angiogenesis model. [•] Comparison of synthesized nanoparticle to non-targeted nanoparticle. [•] Passive accumulation of both nanoparticles was observed in the angiogenesis model. [Copyright &y& Elsevier]

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]

Highlights: [•] Heteronuclear polarization transfer by adiabatic RESPIRATIONCP. [•] Increased transfer efficiency and robustness towards rf inhomogeneity. [•] Demonstrated improved performance for 13C→ 15N polarization transfer in proteins. [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: We present a novel sampling strategy, interleaving acquisition of multiple NMR spectra by exploiting initial polarization subsequently from 1H and 2H spins, taking advantage of their different T 1 relaxation times. Different 1H- and 2H-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) 1H→ 13C/2H→ 13C CP/MAS multiple-acquisition method is demonstrated by 1D and 2D experiments using a uniformly 2H, 15N,13C-labeled α-spectrin SH3 domain sample with all or 30% back-exchanged labile 2H to 1H. It is demonstrated how 1D 13C CP/MAS or 2D 13C–13C correlation spectra initialized with polarization from either 1H or 2H may be recorded simultaneously with flexibility to be added or used individually for spectral editing. It is also shown how 2D 13C–13C correlation spectra may be recorded interleaved with 2H–13C correlation spectra to obtain 13C–13C correlations along with information about dynamics from 2H sideband patterns. [Copyright &y& Elsevier]

Abstract: Serine proteases are classical objects for studies of catalytic and inhibitory mechanisms as well as interesting as therapeutic targets. Since small-molecule serine protease inhibitors generally suffer from specificity problems, peptidic inhibitors, isolated from phage-displayed peptide libraries, have attracted considerable attention. Here, we have investigated the mechanism of binding of peptidic inhibitors to serine protease targets. Our model is upain-1 (CSWRGLENHRMC), a disulfide-bond-constrained competitive inhibitor of human urokinase-type plasminogen activator with a noncanonical inhibitory mechanism and an unusually high specificity. Using a number of modified variants of upain-1, we characterised the upain-1–urokinase-type plasminogen activator complex using X-ray crystal structure analysis, determined a model of the peptide in solution by NMR spectroscopy, and analysed binding kinetics and thermodynamics by surface plasmon resonance and isothermal titration calorimetry. We found that upain-1 changes both main-chain conformation and side-chain orientations as it binds to the protease, in particular its Trp3 residue and the surrounding backbone. The properties of upain-1 are strongly influenced by the addition of three to four amino acids long N-terminal and C-terminal extensions to the core, disulfide-bond-constrained sequence: The C-terminal extension stabilises the solution structure compared to the core peptide alone, and the protease-bound structure of the peptide is stabilised by intrapeptide contacts between the N-terminal extension and the core peptide around Trp3. These results provide a uniquely detailed description of the binding of a peptidic protease inhibitor to its target and are of general importance in the development of peptidic inhibitors with high specificity and new inhibitory mechanisms. [Copyright &y& Elsevier]

Abstract: The folding and stabilization of α-helical transmembrane proteins are still not well understood. Following cofactor binding to a membrane protein provides a convenient method to monitor the formation of appropriate native structures. We have analyzed the assembly and stability of the transmembrane cytochrome b 559′, which can be efficiently assembled in vitro from a heme-binding PsbF homo-dimer by combining free heme with the apo-cytochrome b 559′. Unfolding of the protein dissolved in the mild detergent dodecyl maltoside may be induced by addition of SDS, which at high concentrations leads to dimer dissociation. Surprisingly, absorption spectroscopy reveals that heme binding and cytochrome formation at pH 8.0 are optimal at intermediate SDS concentrations. Stopped-flow kinetics revealed that genuine conformational changes are involved in heme binding at these SDS concentrations. GPS (Global Protein folding State mapping) NMR measurements showed that optimal heme binding is intimately related to a change in the degree of histidine protonation. In the absence of SDS, the pH curve for heme binding is bell-shaped with an optimum at around pH 6–7. At alkaline pH values, the negative electrostatic potential of SDS lowers the local pH sufficiently to restore efficient heme binding, provided the amount of SDS needed for this does not denature the protein. Accordingly, the higher the pH value above 6–7, the more SDS is needed to improve heme binding, and this competes with the inherent tendency of SDS to dissociate cytochrome b 559′. Our work highlights that, in addition to its denaturing properties, SDS can affect protein functions by lowering the local pH. [Copyright &y& Elsevier]

Abstract: Aimed at selective detection of 23Na with specific quadrupolar couplings for in vitro NMR and MRI, we present a series of quadrupolar binomial pulse sequences offering high specificity with respect to the quadrupolar couplings of the excited species. It is demonstrated that pulse sequences with an increasing number of elements, e.g., 11, 121, 1331, 14641, and 15101051, with the units representing flip angles smaller than the 90° pulses typically encountered in binomial spin-1/2 solvent suppression experiments, and different phase combinations may provide a high degree of flexibility with respect to quadrupolar coupling selectivity and robustness towards rf inhomogeneity. This may facilitate efficient separation of, for example, intra and extracellular 23Na in tissues with efficient control of the excitation (or suppression) of central as well as satellite transitions through on- and off-resonance irradiation. The pulse sequences are described in terms of their analogy to binomial liquid-state NMR solvent suppression experiments and demonstrated numerically and experimentally through NMR and MRI experiments on a 7T horizontal small-bore animal magnet system. [ABSTRACT FROM AUTHOR]

Abstract: Due to its unique sensitivity to tissue microstructure, diffusion-weighted magnetic resonance imaging (MRI) has found many applications in clinical and fundamental science. With few exceptions, a more precise correspondence between physiological or biophysical properties and the obtained diffusion parameters remain uncertain due to lack of specificity. In this work, we address this problem by comparing diffusion parameters of a recently introduced model for water diffusion in brain matter to light microscopy and quantitative electron microscopy. Specifically, we compare diffusion model predictions of neurite density in rats to optical myelin staining intensity and stereological estimation of neurite volume fraction using electron microscopy. We find that the diffusion model describes data better and that its parameters show stronger correlation with optical and electron microscopy, and thus reflect myelinated neurite density better than the more frequently used diffusion tensor imaging (DTI) and cumulant expansion methods. Furthermore, the estimated neurite orientations capture dendritic architecture more faithfully than DTI diffusion ellipsoids. [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]

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]

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]

Software facilitating numerical simulation of solid-state NMR experiments on polypeptides is presented. The Tcl-controlled SIMMOL program reads in atomic coordinates in the PDB format from which it generates typical or user-defined parameters for the chemical shift, J coupling, quadrupolar coupling, and dipolar coupling tensors. The output is a spin system file for numerical simulations, e.g., using SIMPSON (Bak, Rasmussen, and Nielsen, J. Magn. Reson.147, 296 (2000)), as well as a 3D visualization of the molecular structure, or selected parts of this, with user-controlled representation of relevant tensors, bonds, atoms, peptide planes, and coordinate systems. The combination of SIMPSON and SIMMOL allows straightforward simulation of the response of advanced solid-state NMR experiments on typical nuclear spin interactions present in polypeptides. Thus, SIMMOL may be considered a “sample changer” to the SIMPSON “computer spectrometer” and proves to be very useful for the design and optimization of pulse sequences for application on uniformly or extensively isotope-labeled peptides where multiple-spin interactions need to be considered. These aspects are demonstrated by optimization and simulation of novel DCP and C7 based 2D N(CO)CA, N(CA)CB, and N(CA)CX MAS correlation experiments for multiple-spin clusters in ubiquitin and by simulation of PISA wheels from PISEMA spectra of uniaxially oriented bacteriorhodopsin and rhodopsin under conditions of finite RF pulses and multiple spin interactions. [Copyright &y& Elsevier]