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The difficulties in quantitatively modeling the temperature dependence of spin–lattice relaxation in a model isotope-enriched peptide are explored as a prelude to obtaining dynamics parameters for motions in proteins from such measurements. The degree to which this can be handled by adding spin diffusion to a bath in standard rate matrix relaxation theory is studied using a small tri-peptide model system, glycyl–alanyl-leucine (GAL). We observe in this molecule that the relaxation of backbone carbons CO and Cα is not dominated by local fluctuations of the 13C–1H dipolar couplings, but rather by 13C–13C spin diffusion to nearby methyl relaxation sinks. A treatment of the methyl relaxation itself, which ignores 13C–13C spin diffusion effects back to the otherwise slowly relaxing bath, provides poor agreement between theory and experimental data obtained for the temperature dependence of the methyl relaxation rates. Closed form approximate spectral densities and relaxation rates for a methyl group during magic angle spinning are obtained to compute the needed transition rates. These average computed rates, in conjunction with an extended form of the Solomon equations, are found to adequately model the temperature dependence of the methyl relaxation rates when spin diffusion is included. The barrier to rotation for the alanine methyl in GAL is determined to be 3.5 kcal mol−1. [ABSTRACT FROM AUTHOR]

The assignment of protein backbone and side-chain NMR chemical shifts is the first step towards the characterization of protein structure. The recent introduction of proton detection in combination with fast MAS has opened up novel opportunities for assignment experiments. However, typical 3D sequential-assignment experiments using proton detection under fast MAS lead to signal intensities much smaller than the theoretically expected ones due to the low transfer efficiency of some of the steps. Here, we present a selective 3D experiment for deuterated and (amide) proton back-exchanged proteins where polarization is directly transferred from backbone nitrogen to selected backbone or sidechain carbons. The proposed pulse sequence uses only 1H-15N cross-polarization (CP) transfers, which are, for deuterated proteins, about 30% more efficient than 1H-13C CP transfers, and employs a dipolar version of the INEPT experiment for N-C transfer. By avoiding HN-C (HN stands for amide protons) and C-C CP transfers, we could achieve higher selectivity and increased signal intensities compared to other pulse sequences containing long-range CP transfers. The REDOR transfer is designed with an additional selective π pulse, which enables the selective transfer of the polarization to the desired 13C spins. [ABSTRACT FROM AUTHOR]

We have previously shown that Congo red (CR) binds site specifically to amyloid fibrils formed by HET-s(218-289) with the long axis of the CR molecule almost parallel to the fibril axis. HADDOCK docking studies indicated that CR adopts a roughly planar conformation with the torsion angle ϕ characterizing the relative orientation of the two phenyl rings being a few degrees. In this study, we experimentally determine the torsion angle ϕ at the center of the CR molecule when bound to HET-s(218-289) amyloid fibrils using solid-state NMR tensor-correlation experiments. The method described here relies on the site-specific C labeling of CR and on the analysis of the two-dimensional magic-angle spinning tensor-correlation spectrum of C-CR. We determined the torsion angle ϕ to be 19°. [ABSTRACT FROM AUTHOR]

We demonstrate measurement of non-equilibrium backbone amide hydrogen-deuterium exchange rates (HDX) for solid proteins. The target of this study are the slowly exchanging residues in solid samples, which are associated with stable secondary-structural elements of proteins. These hydrogen exchange processes escape methods measuring equilibrium exchange rates of faster processes. The method was applied to a micro-crystalline preparation of the SH3 domain of chicken α-spectrin. Therefore, from a 100% back-exchanged micro-crystalline protein preparation, the supernatant buffer was exchanged by a partially deuterated buffer to reach a final protonation level of approximately 20% before packing the sample in a 1.3 mm rotor. Tracking of the HN peak intensities for 2 weeks reports on site-specific hydrogen bond strength and also likely reflects water accessibility in a qualitative manner. H/D exchange can be directly determined for hydrogen-bonded amides using H detection under fast magic angle spinning. This approach complements existing methods and provides the means to elucidate interesting site-specific characteristics for protein functionality in the solid state. [ABSTRACT FROM AUTHOR]

Ice, in addition to 'liquid' water and protein, is an important component of protein samples for NMR spectroscopy at subfreezing temperatures but it has rarely been observed spectroscopically in this context. We characterize its spectroscopic behavior in the temperature range from 100 to 273 K, and find that it behaves like pure water ice. The interference of magic-angle spinning (MAS) as well as rf multiple-pulse sequences with Bjerrum-defect motion greatly influences the ice spectra. [ABSTRACT FROM AUTHOR]

Multiple-bond carbon-carbon homonuclear mixing is a hurdle in extensively deuterated proteins and under fast MAS due to the absence of an effective proton dipolar-coupling network. Such conditions are now commonly employed in solid-state NMR spectroscopy. Here, we introduce an isotropic homonuclear C-C through-bond mixing sequence, MOCCA, for the solid state. Even though applied under MAS, this scheme performs without rotor synchronization and thus does not pose the usual hurdles in terms of power dissipation for fast spinning. We compare its performance with existing homonuclear C-C mixing schemes using a perdeuterated and partially proton-backexchanged protein. Based on the analysis of side chain carbon-carbon correlations, we show that particularly MOCCA with standard 180-degree pulses and delays leading to non-rotor-synchronized spacing performs exceptionally well. This method provides high magnetization transfer efficiency for multiple-bond transfer in the aliphatic region compared with other tested mixing sequences. In addition, we show that this sequence can also be tailor-made for recoupling within a selected spectral region using band-selective pulses. [ABSTRACT FROM AUTHOR]

We discuss the optimum experimental conditions to obtain assignment spectra for solid proteins at magic-angle spinning (MAS) frequencies around 100 kHz. We present a systematic examination of the MAS dependence of the amide proton T′ times and a site-specific comparison of T′ at 93 kHz versus 60 kHz MAS frequency. A quantitative analysis of transfer efficiencies of building blocks, as they are used for typical 3D experiments, was performed. To do this, we compared dipolar-coupling and J-coupling based transfer steps. The building blocks were then combined into 3D experiments for sequential resonance assignment, where we evaluated signal-to-noise ratio and information content of the different 3D spectra in order to identify the best assignment strategy. Based on this comparison, six experiments were selected to optimally assign the model protein ubiquitin, solely using spectra acquired at 93 kHz MAS. Within 3 days of instrument time, the required spectra were recorded from which the backbone resonances have been assigned to over 96 %. [ABSTRACT FROM AUTHOR]

The human voltage dependent anion channel 1 (VDAC) is a 32 kDa β-barrel integral membrane protein that controls the transport of ions across the outer mitochondrial membrane. Despite the determination of VDAC solution and diffraction structures, a structural basis for the mechanism of its function is not yet fully understood. Biophysical studies suggest VDAC requires a lipid bilayer to achieve full function, motivating the need for atomic resolution structural information of VDAC in a membrane environment. Here we report an essential step toward that goal: extensive assignments of backbone and side chain resonances for VDAC in DMPC lipid bilayers via magic angle spinning nuclear magnetic resonance (MAS NMR). VDAC reconstituted into DMPC lipid bilayers spontaneously forms two-dimensional lipid crystals, showing remarkable spectral resolution (0.5-0.3 ppm for C line widths and <0.5 ppm N line widths at 750 MHz). In addition to the benefits of working in a lipid bilayer, several distinct advantages are observed with the lipid crystalline preparation. First, the strong signals and sharp line widths facilitated extensive NMR resonance assignments for an integral membrane β-barrel protein in lipid bilayers by MAS NMR. Second, a large number of residues in loop regions were readily observed and assigned, which can be challenging in detergent-solubilized membrane proteins where loop regions are often not detected due to line broadening from conformational exchange. Third, complete backbone and side chain chemical shift assignments could be obtained for the first 25 residues, which comprise the functionally important N-terminus. The reported assignments allow us to compare predicted torsion angles for VDAC prepared in DMPC 2D lipid crystals, DMPC liposomes, and LDAO-solubilized samples to address the possible effects of the membrane mimetic environment on the conformation of the protein. Concluding, we discuss the strengths and weaknesses of the reported assignment approach and the great potential for even more complete assignment studies and de novo structure determination via H detected MAS NMR. [ABSTRACT FROM AUTHOR]

A considerable limitation of NMR spectroscopy is its inherent low sensitivity. Approximately 90 % of the measuring time is used by the spin system to return to its Boltzmann equilibrium after excitation, which is determined by H-T in cross-polarized solid-state NMR experiments. It has been shown that sample doping by paramagnetic relaxation agents such as Cu-EDTA accelerates this process considerably resulting in enhanced sensitivity. Here, we extend this concept to Gd-complexes. Their effect on H-T has been assessed on the membrane protein proteorhodopsin, a 7TM light-driven proton pump. A comparison between Gd-DOTA, Gd-TTAHA, covalently attached Cu-EDTA-tags and Cu-EDTA reveals a 3.2-, 2.6-, 2.4- and 2-fold improved signal-to-noise ratio per unit time due to longitudinal paramagnetic relaxation enhancement. Furthermore, Gd-DOTA shows a remarkably high relaxivity, which is 77-times higher than that of Cu-EDTA. Therefore, an order of magnitude lower dopant concentration can be used. In addition, no line-broadening effects or peak shifts have been observed on proteorhodopsin in the presence of Gd-DOTA. These favourable properties make it very useful for solid-state NMR experiments on membrane proteins. [ABSTRACT FROM AUTHOR]

Solid-state Nuclear Magnetic Resonance can provide detailed insight into structural and dynamical aspects of complex biomolecules. With increasing molecular size, advanced approaches for spectral simplification and the detection of medium to long-range contacts become of critical relevance. We have analyzed the protonation pattern of a membrane-embedded ion channel that was obtained from bacterial expression using protonated precursors and DO medium. We find an overall reduction of 50% in protein protonation. High levels of deuteration at H and H positions reduce spectral congestion in (H,C,N) correlation experiments and generate a transfer profile in longitudinal mixing schemes that can be tuned to specific resonance frequencies. At the same time, residual protons are predominantly found at amino-acid side-chain positions enhancing the prospects for obtaining side-chain resonance assignments and for detecting medium to long-range contacts. Fractional deuteration thus provides a powerful means to aid the structural analysis of complex biomolecules by solid-state NMR. [ABSTRACT FROM AUTHOR]

An approach for generating efficient $$ {\rm{RN}}_{n}^{\nu_{\rm{S}}, {\nu_{\rm{k}}}} $$ symmetry-based dual channel RF pulse schemes for γ-encoded broadband 15N–13C dipolar recoupling at high magic angle spinning frequencies is presented. The method involves the numerical optimisation of the RF phase-modulation profile of the basic “ R” element so as to obtain heteronuclear double quantum dipolar recoupling sequences with satisfactory magnetisation transfer characteristics. The basic “ R” element was implemented as a sandwich of a small number of short pulses of equal duration with each pulse characterised by a RF phase and amplitude values. The performance characteristics of the sequences were evaluated via numerical simulations and 15N–13C chemical shift correlation experiments. Employing such 13C–15N double-quantum recoupling sequences and the multiple receiver capabilities available in the current generation of NMR spectrometers, the possibility to simultaneously acquire 3D NCC and CNH chemical shift correlation spectra is also demonstrated. [ABSTRACT FROM AUTHOR]

An approach for the efficient implementation of RN symmetry-based pulse schemes that are often employed for recoupling and decoupling of nuclear spin interactions in biological solid state NMR investigations is demonstrated at high magic-angle spinning frequencies. RF pulse sequences belonging to the RN symmetry involve the repeated application of the pulse sandwich { Rϕ R−ϕ}, corresponding to a propagator URF = exp(−i4ϕ Iz), where ϕ = πν/ N and R is typically a pulse that rotates the nuclear spins through 180° about the x-axis . In this study, broadband, phase-modulated 180° pulses of constant amplitude were employed as the initial ‘ R’ element and the phase-modulation profile of this ‘ R’ element was numerically optimised for generating RN symmetry-based pulse schemes with satisfactory magnetisation transfer characteristics. At representative MAS frequencies, RF pulse sequences were implemented for achieving 13C–13C double-quantum dipolar recoupling and through bond scalar coupling mediated chemical shift correlation and evaluated via numerical simulations and experimental measurements. The results from these investigations are presented here. [ABSTRACT FROM AUTHOR]

The CN class of RF pulse schemes, commonly employed for recoupling and decoupling of nuclear spin interactions in magic angle spinning solid state NMR studies of biological systems, involves the application of a basic “ C” element corresponding to an RF cycle with unity propagator. In this study, the design of CN symmetry-based RF pulse sequences for achieving 13C–13C double-quantum dipolar recoupling and through bond scalar coupling mediated 13C–13C chemical shift correlation has been examined at high MAS frequencies employing broadband, constant-amplitude, phase-modulated basic “ C” elements. The basic elements were implemented as a sandwich of a small number of short pulses of equal duration with each pulse characterised by an RF phase value. The phase-modulation profile of the “ C” element was optimised numerically so as to generate efficient RF pulse sequences. The performances of the sequences were evaluated via numerical simulations and experimental measurements and are presented here. [ABSTRACT FROM AUTHOR]

We demonstrate that short, medium and long-range constraints can be extracted from proton mediated, rare-spin detected correlation solid-state NMR experiments for the microcrystalline 10.4 × 2 kDa dimeric model protein Crh. Magnetization build-up curves from cross signals in NHHC and CHHC spectra deliver detailed information on side chain conformers and secondary structure for interactions between spin pairs. A large number of medium and long-range correlations can be observed in the spectra, and an analysis of the resolved signals reveals that the constraints cover the entire sequence, also including inter-monomer contacts between the two molecules forming the domain-swapped Crh dimer. Dynamic behavior is shown to have an impact on cross signals intensities, as indicated for mobile residues or regions by contacts predicted from the crystal structure, but absent in the spectra. Our work validates strategies involving proton distance measurements for large and complex proteins as the Crh dimer, and confirms the magnetization transfer properties previously described for small molecules in solid protein samples. [ABSTRACT FROM AUTHOR]

Solid-state NMR (ssNMR) provides increasing possibilities to study structure and dynamics of biomolecular systems. Our group has been interested in developing ssNMR-based approaches that are applicable to biomolecules of increasing molecular size and complexity without the need of specific isotope-labelling. Methodological aspects ranging from spectral assignments to the indirect detection of proton–proton contacts in multi-dimensional ssNMR are discussed and applied to (membrane) protein complexes. [ABSTRACT FROM AUTHOR]

At high magic angle spinning (MAS) frequencies the potential of tanh/tan adiabatic RF pulse schemes for 13C chemical shift correlation without 1H decoupling during mixing has been evaluated. It is shown via numerical simulations that a continuous train of adiabatic 13C inversion pulses applied at high RF field strengths leads to efficient broadband heteronuclear decoupling. It is demonstrated that this can be exploited effectively for generating through-bond and through-space, including double-quantum, correlation spectra of biological systems at high magnetic fields and spinning speeds with no 1H decoupling applied during the mixing period. Experiments carried out on a polycrystalline sample of histidine clearly suggest that an improved signal to noise ratio can be realised by eliminating 1H decoupling during mixing. [ABSTRACT FROM AUTHOR]

Biophysical studies on membrane proteins by solid state NMR (SSNMR) can be carried out directly in a membrane environment. Samples are usually prepared in form of multi-lamellar dispersions for magic angle sample spinning or as aligned multi-layers for orientation dependent NMR experiments without sample rotation. A new development is the application of MAS NMR to aligned samples (MAOSS; Magic Angle Oriented Sample Spinning). In combination with separated local field (SLF) experiments, size and orientation of heteronuclear dipolar couplings may be extracted from two-dimensional experiments which correlate dipolar couplings with isotropic chemical shifts. The orientation of these 1H–X dipolar couplings can be directly related to the orientation of molecular groups in the sample. Here, we demonstrate the feasibility of these experiments on highly ordered polyethylene fibers which serve as model compound. Based on these data, the experiment is also applied to ordered multi-layers of bacteriorhodopsin (purple membrane) which is used as a model for aligned membrane proteins. We present a detailed analysis of different experimental designs with respect to angular sensitivity and the influence of residual sample disorder (“mosaic spread”). The results of the MAOSS-SLF experiment are discussed within the context of established solid state NMR experiments which are usually performed without sample rotation and we compare the data to orientation information obtained from X-ray diffraction. [ABSTRACT FROM AUTHOR]

The efficacy of hetero- and homonuclear dipolar recoupling employing tanh/tan adiabatic inversion pulse based RF pulse schemes has been examined at high magic angle spinning (MAS) frequencies via numerical simulations and experimental measurements. An approach for minimising the recoupling RF power level is presented, taking into consideration the spinning speed, the range of resonance offsets and H1 inhomogeneities and the available RF field strength. This involves the tailoring of the frequency and amplitude modulation profiles of the inversion pulses. The applicability of tanh/tan pulse based dipolar recoupling schemes to spinning speed regimes where the performance with conventional rectangular pulses may not be satisfactory is demonstrated. [ABSTRACT FROM AUTHOR]

A simple approach is demonstrated for designing optimised broadband inversion pulses for MAS solid state NMR studies of biological systems. The method involves a two step numerical optimisation procedure and takes into account experimental requirements such as the pulse length, resonance offset range and extent of H1 inhomogeneity compensation needed. A simulated annealing protocol is used initially to find appropriate values for the parameters that define the well known tanh/tan adiabatic pulse such that a satisfactory spin inversion is achieved with minimum RF field strength. This information is then used in the subsequent stage of refinement where the RF pulse characteristics are further tailored via a local optimisation procedure without imposing any restrictions on the amplitude and frequency modulation profiles. We demonstrate that this approach constitutes a generally applicable tool for obtaining pulses with good inversion characteristics. At moderate MAS frequencies the efficacy of the method is experimentally demonstrated for generating double-quantum NMR spectra via the zero-quantum dipolar recoupling scheme RFDR. [ABSTRACT FROM AUTHOR]

We describe the simplification of 13C–13C correlation spectra obtained from a microcrystalline protein sample expressed on a growth medium of 10% fully 13C labeled glucose diluted in 90% natural abundance glucose as compared to a fully labeled sample. Such a labeling scheme facilitates the backbone and side-chain resonance assignment of Phe, Tyr, His, Asp, Asn, Ile, Lys and Pro and yields an unambiguous stereospecific assignment of the valine Cγ1, Cγ2 13C resonances and of Leucine Cδ2. [ABSTRACT FROM AUTHOR]

Abstract [ABSTRACT FROM AUTHOR]

The efficacy of RN? n symmetry-based adiabatic Zero-Quantum (ZQ) dipolar recoupling schemes for obtaining chemical shift correlation data at moderate magic angle spinning frequencies has been evaluated. RNsequences generally employ basic inversion elements that correspond to a net 180° rotation about the rotating framex-axis. It is shown here via numerical simulations and experimental measurements that it is also possible to achieve efficient ZQ dipolar recoupling via RNschemes employing adiabatic pulses. Such an approach was successfully used for obtaining13C chemical shift correlation spectra of a uniformly labelled sample of (CUG)97- a triplet repeat expansion RNA that has been implicated in the neuromuscular disease myotonic dystrophy. An analysis of the13C sugar carbon chemical shifts suggests, in agreement with our recent15N MAS-NMR studies, that this RNA adopts an A-helical conformation. [ABSTRACT FROM AUTHOR]

One and two-dimensional solid-state NMR experiments are discussed that permit probing local structure and overall molecular conformation of membrane-embedded polypeptides under Magic Angle Spinning. The functional dependence of a series of anisotropic recoupling schemes is analyzed using theoretical and numerical methods. These studies lead to the construction of a set of polarization dephasing or transfer units that probe local backbone conformation and overall molecular orientation within the same NMR experiment. Experimental results are shown for a randomly oriented peptide and for two model membrane-peptides reconstituted into lipid bilayers and oriented on polymer films according to a method proposed by Bechinger etal. [J. Am. Chem. Soc.,124, (2002), 1146-1147]. [ABSTRACT FROM AUTHOR]

Solid state NMR sample preparation and resonance assignments of the U-[13C,15N] 2×10.4 kDa dimeric form of the regulatory protein Crh in microcrystalline, PEG precipitated form are presented. Intra– and interresidue correlations using dipolar polarization transfer methods led to nearly complete sequential assignments of the protein, and to 88% of all 15N, 13C chemical shifts. For several residues, the resonance assignments differ significantly from those reported for the monomeric form analyzed by solution state NMR. Dihedral angles obtained from a TALOS-based statistical analysis suggest that the microcrystalline arrangement of Crh must be similar to the domain-swapped dimeric structure of a single crystal form recently solved using X-ray crystallography. For a limited number of protein residues, a remarkable doubling of the observed NMR resonances is observed indicative of local static or dynamic conformational disorder. Our study reports resonance assignments for the largest protein investigated by solid state NMR so far and describes the conformational dimeric variant of Crh with previously unknown chemical shifts. [ABSTRACT FROM AUTHOR]

2D slow-spinning, rotor-synchronized MAS exchange spectroscopy (SSRS-MASE) was applied to study local secondary structure of three structurally different peptides, two of which were membrane-bound. Each peptide was 13C carbonyl labeled at two adjacent residues in the peptide backbone. In general, this methodology is attractive for membrane-bound peptides because of its lenient spinning, decoupling, and RF homogeneity requirements. For a single set of raw SSRS-MASE data, two linearly independent methods exist for obtaining a 2D spectrum and each spectrum can be fit to obtain conformational constraints. An approach is described for combining the results of these two fits and this method is shown to work for spectra with both resolved and unresolved labeled site resonances. A spectrum is often fit well to a few different conformations which have somewhat different values of the fitting parameter χ2. A simple statistical theory is developed which relates the Δχ2 difference between a local minimum and the global minimum χ2 to the likelihood that the local minimum conformation is the correct structure. Because uncertainty in the simulated data can also contribute to the overall fitting uncertainty, an empirical method is described for incorporating the simulation uncertainty into the Δχ2 analysis. These data analysis methods were tested on polycrystalline Ala-Gly-Gly and then applied to the membrane-bound melittin and HIV-1 fusion peptides. Melittin gave a best-fit α helical structure at Ala-4 while the fusion peptide gave a good-fit β strand structure at Phe-8. The melittin analysis is in agreement with the known overall structure of this peptide. [ABSTRACT FROM AUTHOR]

The application of adiabatic polarization-transfer experiments to resonance assignment in solid, uniformly 13C-15N-labelled polypeptides is demonstrated for the cyclic decapeptide antamanide. A homonuclear correlation experiment employing the DREAM sequence for adiabatic dipolar transfer yields a complete assignment of the Cα and aliphatic side-chain 13C resonances to amino acid types. The same information can be obtained from a TOBSY experiment using the recently introduced P9112 TOBSY sequence, which employs the J couplings as a transfer mechanism. A comparison of the two methods is presented. Except for some aromatic phenylalanine resonances, a complete sequence-specific assignment of the 13C and 15N resonances in antamanide is achieved by a series of selective or broadband adiabatic triple-resonance experiments. Heteronuclear transfer by adiabatic-passage Hartmann–Hahn cross polarization is combined with adiabatic homonuclear transfer by the DREAM and rotational-resonance tickling sequences into two- and three-dimensional experiments. The performance of these experiments is evaluated quantitatively. [ABSTRACT FROM AUTHOR]

One- and two-dimensional solid-state NMR experiments on a uniformly labeled intrinsic membrane-protein complex at ultra-high magnetic fields are presented. Two-dimensional backbone and side-chain correlations for a [U-13C,15N] labeled version of the LH2 light-harvesting complex indicate significant resolution at low temperatures and under Magic Angle Spinning. Tentative assignments of some of the observed correlations are presented and attributed to the α-helical segments of the protein, mostly found in the membrane interior. [ABSTRACT FROM AUTHOR]

An approach to the determination of the orientation of the carbonyl chemical shift (CS) tensor in a 13C′-15N-1H dipolar coupled spin network is proposed. The method involves the measurement of the Euler angles of the 13C′-15N and 15N-1H dipolar vectors in the 13C′ CS tensor principal axes system, respectively, via a 13C-15N REDOR experiment and by a 2D relayed anisotropy correlation of the 13C′ CSA (ω2) and 15N-1H dipolar interaction (ω1). Via numerical simulations the sensitivity of the ω1 cross sections of the 2D spectrum to the Euler angles of the 15N-1H bond vector in the 13C′ CSA frame is shown. Employing the procedure outlined in this work, we have determined the orientation of the 13C′ CS tensor in the peptide plane of the dipeptide AibAib-NH2 (Aib = α-aminoisobutyric acid). The Euler angles are found to be (χCN, ψCN) = (34° ± 2°, 88° ± 2° ) and (χNH, ψNH) = (90° ± 10°, 80° ± 10° ). From the measured Euler angles it is seen that the σ33 and σ22 components of the 13C′ CS tensor approximately lie in the peptide plane. [ABSTRACT FROM AUTHOR]

An approach to the determination of the 2-13C′ chemical shift (CS) tensor orientation in pyrimidine bases via heteronuclear MAS NMR spectroscopy is presented. Considering a dipolar coupled spin 1/2 network of the type S1-I-S2 consisting of directly bonded heteronuclear spins, we have carried out numerical simulations to assess the sensitivity of I-S REDOR spinning sidebands to the Euler angles defining the orientation of the I-S1 and I-S2 dipolar vectors in the I spin CS tensor principal axes system. Our investigations clearly demonstrate the potential of I-S REDOR studies in IS1S2 systems for obtaining with high reliability and accuracy the I spin chemical shift tensor orientation in the molecular frame spanned by the two internuclear vectors I-S1 and I-S2. The significant contribution to the observed REDOR sideband intensities from anti-phase operator terms which are present at the start of the data acquisition is illustrated. The procedure for the recording and analysis of the I-S REDOR spectra in IS1S2 systems is presented and the measurement of the 2-13C′ CS tensor orientation in a polycrystalline sample of [1,3-15N2, 2-13C] uracil, which is one of the four bases in RNA, is experimentally demonstrated. [ABSTRACT FROM AUTHOR]

The complete three-dimensional (3D) structure of a glycylisoleucine (Gly-Ile) molecule was determined by individually measuring six dihedral angles with a frequency-selective homonuclear dipolar recoupling method, R2TR (rotational resonance in the tilted rotating frame), using a powder sample of diluted uniformly 13,15-labeled Gly-Ile. Each dihedral angle was obtained by recoupling a dipolar interaction between three or four bonds distant spins concerned or observing a dipolar correlation 2D powder pattern. The 3D structure of a Gly-Ile molecule was also determined by X-ray crystallography, and a good agreement with the NMR result was obtained. The results demonstrate that the R2TR method in a uniformly labeled powder sample can provide the 3D structure without the need to prepare a lot of selectively labeled samples. [ABSTRACT FROM AUTHOR]