Double Cross-polarization Research Articles

Satellite-transition double cross-polarization HETCOR under fast MAS

Double-cross polarization to the satellite-transitions (STs) of half-integer quadrupolar nuclei is demonstrated using proton-detected heteronuclear correlation (HETCOR) under fast magic-angle spinning (MAS). By placing the rf frequency away from the central-transition (CT) and selective to the STs, average Hamiltonian theory shows a scaled effective rf field with a phase equal to the complex ST spinning sideband being irradiated. Such an effective rf field can excite and spinlock STs but the phase spread usually leads to signal cancellation in one-step excitation or cross polarization experiments. The cancellation does not occur for two-step double cross-polarization (DCP) HETCOR experiments, therefore high efficiencies can be obtained. With careful magic-angle calibration, ST and double-quantum ST (DQST) HETCOR experiments are demonstrated with the 35Cl nuclei in histidine·HCl·H2O. These experiments provide additional information over the commonly observed CT spectra and near isotropic resolution in the case of DQST of spin S = 3/2 quadrupolar nuclei.

77Se-13C based dipolar correlation experiments to map selenium sites in microcrystalline proteins.

Sulfur-containing sites in proteins are of great importance for both protein structure and function, including enzymatic catalysis, signaling pathways, and recognition of ligands and protein partners. Selenium-77 is an NMR active spin-1/2 nucleus that shares many physiochemical properties with sulfur and can be readily introduced into proteins at sulfur sites without significant perturbations to the protein structure. The sulfur-containing amino acid methionine is commonly found at protein-protein or protein-ligand binding sites. Its selenium-containing counterpart, selenomethionine, has a broad chemical shift dispersion useful for NMR-based studies of complex systems. Methods such as (1H)-77Se-13C double cross polarization or {77Se}-13C REDOR could be valuable to map the local environment around selenium sites in proteins but have not been demonstrated to date. In this work, we explore these dipolar transfer mechanisms for structural characterization of the GB1 V39SeM variant of the model protein GB1 and demonstrate that 77Se-13C based correlations can be used to map the local environment around selenium sites in proteins. We have found that the general detection limit is ~ 5Å, but longer range distances up to ~ 7Å can be observed as well. This study establishes a framework for the future characterization of selenium sites at protein-protein or protein-ligand binding interfaces.

Self-Assembly of DNA and RNA Building Blocks Explored by Nitrogen-14 NMR Crystallography: Structure and Dynamics.

The isotopic enrichment of nucleic acids with nitrogen-15 is often carried out by solid-phase synthesis of oligonucleotides using phosphoramidite precursors that are synthetically demanding and expensive. These synthetic challenges, combined with the overlap of chemical shifts, explain the lag of nitrogen-15 NMR studies of nucleic acids behind those of proteins. For the structural characterization of DNA and RNA-related systems, new NMR methods that exploit the naturally occurring 99.9 % abundant nitrogen-14 isotope are therefore highly desirable. In this study, we have investigated nitrogen-14 spectra of self-assembled quartets based on the nucleobase guanine in the solid state by means of magic-angle spinning NMR spectroscopy. The network of dipolar proton-nitrogen couplings between neighboring stacked purine units is probed by 2D spectra based on 1 H→14 N→1 H double cross-polarization. Interplane dipolar contacts are identified between the stacked G quartets. The assignment is supported by density functional theory (DFT) calculations of the anisotropic chemical shifts and quadrupolar parameters. The experimental spectra are fully consistent with internuclear distances obtained in silico. Averaging of chemical shifts due to internal motions can be interpreted by semiempirical calculations. This method can easily be extended to synthetic G quartets based on nucleobase or nucleoside analogs and potentially to oligonucleotides.

Sensitivity enhancement in 2D Double Cross Polarization experiments under fast MAS by recycling unused protons

We demonstrate sensitivity enhancement via recycling of proton magnetization in 2D Double Cross Polarization (Double CP) experiments performed on fully protonated and uniformly labeled (13C, 15N) samples at a magic angle spinning rate of 60 kHz. Unused proton magnetization is preserved during t1 evolution either by locking it with CW irradiation or by employing rotor-synchronized pi pulses. A flip-back pulse together with a modified second CP block preserves unused proton magnetization resulting in enhanced sensitivity. We have achieved sensitivity enhancements of 15–20% and 25–28% in 1H–13C and 1H–15N 2D Double CP experiments respectively. At shorter recycle delays (∼0.25T1), relative sensitivity enhancements of 40–45% and 55% were obtained in 1H–13C and 1H–15N 2D Double CP experiments respectively. An analysis of the sensitivity enhancements and theoretical estimation of lineshapes in indirect dimension in the presence of proton recycling is provided.

Sensitivity enhancement via multiple contacts in the {1H-29Si}-1H cross polarization experiment: a case study of modified silica nanoparticle surfaces.

{1H–29Si}–1H double cross polarization inverse detection (DCPi) solid-state NMR, has recently been shown to be a powerful tool for studying molecules adsorbed on the silica surface. In this contribution, we develop an improved version (MCPi) which incorporates a block of multiple contact pulses, and quantitatively compare the sensitivities of MCPi and DCPi over a typical range of experimental parameters. The MCPi pulse sequence aims at higher sensitivity and robustness for studying samples with various relaxation characteristics. In the case of dimethyl sulfoxide (DMSO) molecules adsorbed on the silica surface, MCPi performs equally well or up to 2.5 times better than DCPi over a wide range of parameters. The applicability to and performance of MCPi on composite materials was demonstrated using a sample of polymer–silica composite, where significantly higher sensitivity could be achieved at very long total mixing times. The results also showed that both techniques are surface specific in the sense that only the groups close to the surface can be detected.

Sensitivity boosts by the CPMAS CryoProbe for challenging biological assemblies

Despite breakthroughs in MAS NMR hardware and experimental methodologies, sensitivity remains a major challenge for large and complex biological systems. Here, we report that 3–4 fold higher sensitivities can be obtained in heteronuclear-detected experiments, using a novel HCN CPMAS probe, where the sample coil and the electronics operate at cryogenic temperatures, while the sample is maintained at ambient temperatures (BioSolids CryoProbe™). Such intensity enhancements permit recording 2D and 3D experiments that are otherwise time-prohibitive, such as 2D 15N-15N proton-driven spin diffusion and 15N-13C double cross polarization to natural abundance carbon experiments. The benefits of CPMAS CryoProbe-based experiments are illustrated for assemblies of kinesin Kif5b with microtubules, HIV-1 capsid protein assemblies, and fibrils of human Y145Stop and fungal HET-s prion proteins – demanding systems for conventional MAS solid-state NMR and excellent reference systems in terms of spectral quality. We envision that this probe technology will be beneficial for a wide range of applications, especially for biological systems suffering from low intrinsic sensitivity and at physiological temperatures.

Double cross polarization for the indirect detection of nitrogen-14 nuclei in magic angle spinning NMR spectroscopy.

Nitrogen-14 NMR spectra at fast magic-angle spinning rates can be acquired indirectly by means of two-dimensional techniques based on double cross polarization transfer 1H → 14N →1H. Experimental evidence is given for polycrystalline samples of glycine, l-histidine, and the dipeptide Ala-Gly. Either one-bond or long-range correlations can be favored by choosing the length of the cross polarization contact pulses. Longer contact pulses allow the detection of unprotonated nitrogen sites. In contrast to earlier methods that exploited second-order quadrupolar/dipolar cross-terms, cross polarization operates in the manner of the method of Hartmann and Hahn, even for 14N quadrupolar couplings up to 4 MHz. Simulations explain why amorphous samples tend to give rise to featureless spectra because the 14N quadrupolar interactions may vary dramatically with the lattice environment. The experiments are straightforward to set up and are shown to be effective for different nitrogen environments and robust with respect to the rf-field strengths and to the 14N carrier frequency during cross polarization. The efficiency of indirect detection of 14N nuclei by double cross polarization is shown to be similar to that of isotopically enriched 13C nuclei.

Magic angle Lee–Goldburg frequency offset irradiation improves the efficiency and selectivity of SPECIFIC-CP in triple-resonance MAS solid-state NMR

The efficiency and selectivity of SPECIFIC-CP, a widely used method for selective double cross-polarization in triple-resonance magic angle spinning solid-state NMR, is improved by performing the tangential-shaped 13C irradiation at an offset frequency that meets the Lee–Goldburg condition (LG-SPECIFIC-CP). This is demonstrated on polycrystalline samples of uniformly 13C, 15N labeled N-acetyl-leucine and N-formyl-Met-Leu-Phe-OH (MLF) at 700MHz and 900MHz 1H resonance frequencies, respectively. For the single 13Cα of N-acetyl-leucine, relative to conventional broad band cross-polarization, the SPECIFIC-CP signal has 47% of the intensity. Notably, the LG-SPECIFIC-CP signal has 72% of the intensity, essentially the theoretical maximum. There were no other changes in the experimental parameters. The three 13Cα signals in MLF show some variation in intensities, reflecting the relatively narrow bandwidth of a frequency-offset procedure, and pointing to future developments for this class of experiment.

Asymmetric simultaneous phase-inversion cross-polarization in solid-state MAS NMR: Relaxing selective polarization transfer condition between two dilute spins

Double cross polarization (DCP) has been widely used for heteronuclear polarization transfer between 13C and 15N in solid-state magic-angle spinning (MAS) NMR. However, DCP is such sensitive to experimental settings that small variations or deviations in RF fields would deteriorate its efficiency. Here, we report on asymmetric simultaneous phase-inversion cross polarization (referred as aSPICP) for selective polarization transfer between low-γ 13C and 15N spins. We have demonstrated through simulations and experiments using biological solids that the asymmetric duration in the simultaneous phase-inversion cross polarization scheme leads to efficient polarization transfer between 13C and 15N even with large chemical shift anisotropies in the presence of B1 field variations or mismatch of the Hartmann–Hahn conditions. This could be very useful in the aspect of long-duration experiments for membrane protein studies at high fields.

Study of the Mechanism of Thermal Chemical Processes in the Crystals of YAF Tripeptides by Means of Mass Spectrometry and Solid State NMR

Thermal reactions in two Tyr-Ala-Phe (YAF) tripeptide crystals with different molecular packing (monoclinic and hexagonal), distinct stereochemistry of central amino acid (D or L alanine) and specific arrangement of molecules in the crystal lattice (head-to-tail) were investigated. Samples were heated up to 180 °C, while the melting point for YAF crystals is above the 220 °C. Below the melting temperature, in both cases the chemical reactions leading to formation of cyclic dipeptides (YA diketopiperazine) and leaving of phenylalanine were observed. Two possible mechanisms of chemical reaction in the crystal lattice assuming intra- and/or intermolecular pathways were considered. (13)C and (15)N enriched YAF samples were employed to study of mechanism of solid state reactivity using mass spectrometry and advanced solid state NMR techniques (2D DARR (Dipolar Assisted Rotational Resonance) and 2D Double CP (Cross-Polarization) correlations).

Bandwidth in double cross-polarization MAS NMR spectroscopy

The signal intensity in double cross-polarization (DCP) NMR experiments is critically dependent on the experimental parameters, which include the rf field strength, carrier frequency, and magic-angle spinning (MAS) frequency. In this systematic study, we have monitored {1H}/31P/13C DCP signals from monosaccharide α-D-[UL-13C6] galactopyranosyl 1-phosphate (GalP) at a MAS frequency of 13kHz, at which only double quantum cross-polarization (CP) coherence transfer is allowed. To lessen the stringent requirements for these experimental parameters, we have implemented linear ramp pulse, adiabatic ramp-shaped pulse, and block pulse during the period of 31P/13C CP. We unravel the CP matching profiles with respect to these parameters by monitoring the 31P/13C signal while varying the rf field strength and carrier frequency. For comparison, we extracted the selectivity bandwidth from the full width at half maximum (FWHM) of the matching profiles, in units of frequency (kHz), and found bandwidths of 1.1, 14, and 22kHz for the matching profiles of the 13C rf field strength and the 13C and 31P carrier frequencies, respectively, for a linear ramp pulse CP. These bandwidths are broader than the measured values in an adiabatic-shaped pulse CP (0.8, 10, and 12kHz), as well as in block CP (0.3, 7, and 10kHz) experiments. We demonstrate that the linear ramp pulse CP is superior to both block CP and adiabatic-shaped CP in lessening the stringent requirements of the aforementioned experimental settings for DCP experiments.

Double cross-polarization MAS NMR in the assignment of abundant-spin resonances: 19F–{29Si}–19F FBCP/MAS NMR of fluoride ions incorporated in calcium silicate hydrate (C–S–H) phases

A new version of the double cross-polarization MAS NMR experiment, which transfers polarization Forth and Back (FBCP) between high- and low-γ spin nuclei, is presented. The pulse sequence is demonstrated by 19F–{29Si}–19F and 19F–{13C}–19F FBCP NMR spectra of a mixture of cuspidine (Ca4Si2O7F2) and Teflon (–CF2–)n. The experiment is useful for assignment of the high-γ spin resonances, as demonstrated by 19F–{29Si}–19F FBCP NMR of a fluoride-containing calcium–silicate–hydrate (C–S–H) phase, where the 19F resonance from fluoride ions incorporated in the interlayer structure of the C–S–H phase is identified.

Dual-band selective double cross polarization for heteronuclear polarization transfer between dilute spins in solid-state MAS NMR

A sinusoidal modulation scheme is described for selective heteronuclear polarization transfer between two dilute spins in double cross polarization magic-angle-spinning nuclear magnetic resonance spectroscopy. During the second N→C cross polarization, the 13C RF amplitude is modulated sinusoidally while the 15N RF amplitude is tangent. This modulation induces an effective spin-lock field in two selective frequency bands in either side of the 13C RF carrier frequency, allowing for simultaneous polarization transfers from 15N to 13C in those two selective frequency bands. It is shown by experiments and simulations that this sinusoidal modulation allows one to selectively polarize from 15N to its covalently bonded 13Cα and 13C’ carbons in neighboring peptide planes simultaneously, which is useful for establishing the backbone connectivity between two sequential residues in protein structural elucidation. The selectivity and efficiency were experimentally demonstrated on a uniformly 13C,15N-labeled β1 immunoglobulin binding domain of protein G (GB1).

Investigation of the Interface in Silica-Encapsulated Liposomes by Combining Solid State NMR and First Principles Calculations

In the context of nanomedicine, liposils (liposomes and silica) have a strong potential for drug storage and release schemes: such materials combine the intrinsic properties of liposome (encapsulation) and silica (increased rigidity, protective coating, pH degradability). In this work, an original approach combining solid state NMR, molecular dynamics, first principles geometry optimization, and NMR parameters calculation allows the building of a precise representation of the organic/inorganic interface in liposils. {(1)H-(29)Si}(1)H and {(1)H-(31)P}(1)H Double Cross-Polarization (CP) MAS NMR experiments were implemented in order to explore the proton chemical environments around the silica and the phospholipids, respectively. Using VASP (Vienna Ab Initio Simulation Package), DFT calculations including molecular dynamics, and geometry optimization lead to the determination of energetically favorable configurations of a DPPC (dipalmitoylphosphatidylcholine) headgroup adsorbed onto a hydroxylated silica surface that corresponds to a realistic model of an amorphous silica slab. These data combined with first principles NMR parameters calculations by GIPAW (Gauge Included Projected Augmented Wave) show that the phosphate moieties are not directly interacting with silanols. The stabilization of the interface is achieved through the presence of water molecules located in-between the head groups of the phospholipids and the silica surface forming an interfacial H-bonded water layer. A detailed study of the (31)P chemical shift anisotropy (CSA) parameters allows us to interpret the local dynamics of DPPC in liposils. Finally, the VASP/solid state NMR/GIPAW combined approach can be extended to a large variety of organic-inorganic hybrid interfaces.

Operator-based analytic theory of decoherence in NMR

The operator-based analytic description of polarization transfer in NMR spectroscopy is often fraught with difficulty due to (a) the dimension and (b) the non-commuting nature of the spin Hamiltonians. In this article, an analytic model is presented to elucidate the mechanism of polarization transfer between dilute spins I 1 and I 2 coupled to a reservoir of abundant S-spins (i.e. ) in the solid state. Specifically, the factors responsible for the decoherence observed in double cross-polarization (DCP) experiments are outlined in terms of operators via effective Floquet Hamiltonians. The interplay between the various anisotropic interactions is thoroughly investigated by comparing the simulations from the analytic theory with exact numerical methods. The analytical theory presents a framework for incorporating multi-spin effects within a reduced subspace spanned by spins I 1 and I 2. The simulation results from the analytic model comprising eight spins are in excellent agreement with the numerical methods and present an attractive tool for understanding the phenomenon of decoherence in NMR.

A practical guide for the setup of a 1H– 31P– 13C double cross-polarization (DCP) experiment

O-phospho- l-threonine is a convenient sample to setup a 1H– 31P– 13C double cross-polarization (DCP) Hartmann–Hahn match. The 1H– 31P– 13C technique is extremely sensitive to the rate of the sample spinning. Both zero-quantum (ZQ) and double-quantum (DQ) cross-polarization operate at an average spinning rate (6–7 kHz). At higher spinning rates (10 kHz), the DQCP mechanism dominates and leads to a reduction of signal intensity, in particular for lower 31P RF field strength. The application of two shape pulses during the second cross-polarization greatly improves the signal to noise ratio allowing the recording of better quality spectra. 31P– 13C spectrally induced filtering in combination with cross-polarization (SPECIFIC-CP) experiments can be carried out under ZQCP and DQCP conditions if careful attention is paid to the choice of RF field amplitudes and carriers Ω. Application of 1D and 2D 1H– 31P– 13C experiments is demonstrated on model samples; disodium ATP hydrate and O-phospho- l-tyrosine.

Water speciation in hydrous silicate and aluminosilicate glasses: Direct evidence from 29Si-1H and 27Al-1H double-resonance NMR

Through a combination of 1 H MAS NMR, 1 H → 29 Si → 1 H double cross-polarization (CP) MAS NMR and 27 Al → 1 H CP MAS NMR, different OH species [SiOH, AlOH, and (Ca,Mg)OH (free OH)] have been unambiguously identified for hydrous Ca,Mg-(alumino)silicate glasses. This confirms my earlier speciation assignments made partially on the basis of 1 H chemical shift arguments. The dissolution mechanisms of water in both Al-free silicate and aluminosilicate glasses (quenched melts) are fundamentally similar. For relatively polymerized compositions, it involves dominantly the formation of TOH species (T: Si, Al) through the rupture of T-O-T linkages, in addition to molecular H 2 O; for more depolymerized compositions containing network-modifying cations of large field strength (e.g., Ca, Mg), free OH species are also important.

Coherence selection in double CP MAS NMR spectroscopy

Applications of double cross-polarization (CP) magic-angle spinning (MAS) NMR spectroscopy, via 1H/ 15N and then 15N/ 13C coherence transfers, for 13C coherence selection are demonstrated on a 15N/ 13C-labeled N-acetyl-glucosamine (GlcNAc) compound. The 15N/ 13C coherence transfer is very sensitive to the settings of the experimental parameters. To resolve explicitly these parameter dependences, we have systematically monitored the 13C{ 15N/ 1H} signal as a function of the rf field strength and the MAS frequency. The data reveal that the zero-quantum coherence transfer, with which the 13C effective rf field is larger than that of the 15N by the spinning frequency, would give better signal sensitivity. We demonstrate in one- and two-dimensional double CP experiments that spectral editing can be achieved by tailoring the experimental parameters, such as the rf field strengths and/or the MAS frequency.

A High-Resolution43Ca Solid-State NMR Study of the Calcium Sites of Hydroxyapatite

High resolution 43Ca solid-state NMR studies of hydroxyapatite (Ca10(PO4)6(OH)2) were performed at 14.1 T. The two crystallographically distinct calcium sites were unequivocally resolved by a triple-quantum magic angle spinning experiment, and the unambiguous assignment of the signals was possible using 1H-43Ca rotational echo double resonance and 1H-43Ca cross polarization magic angle spinning experiments.

NMR Investigations of the Static and Dynamic Structures of Bisphosphonates on Human Bone: a Molecular Model

We report the results of an investigation of the binding of a series of bisphosphonate drugs to human bone using 2H, 13C, 15N, and 31P nuclear magnetic resonance spectroscopy. The 31P NMR results show that the bisphosphonate groups bind irrotationally to bone, displacing orthophosphate from the bone mineral matrix. Binding of pamidronate is well described by a Langmuir-like isotherm, from which we deduce an approximately 30-38 A2 surface area per pamidronate molecule and a deltaG = -4.3 kcal mol(-1). TEDOR of [13C3, 15N] pamidronate on bone shows that the bisphosphonate binds in a gauche [N-C(1)] conformation. The results of 31P as well as 15N shift and cross-polarization measurements indicate that risedronate binds weakly, since it has a primarily neutral pyridine side chain, whereas zoledronate (with an imidazole ring) binds more strongly, since the ring is partially protonated. The results of 2H NMR measurements of side-chain 2H-labeled pamidronate, alendronate, zoledronate, and risedronate on bone show that all side chains undergo fast but restricted motions. In pamidronate, the motion is well simulated by a gauche+/gauche- hopping motion of the terminal -CH2-NH3(+) group, due to jumps from one anionic surface group to another. The results of double-cross polarization experiments indicate that the NH3(+)-terminus of pamidronate is close to the bone mineral surface, and a detailed model is proposed in which the gauche side-chain hops between two bone PO4(3-) sites.