Hartmann-Hahn Cross-polarization Research Articles

Cross polarization from dipolar-order under magic angle spinning: The ADRF-CPMAS NMR experiment

Techniques for enhancing the signals arising from low-γ, insensitive (I) nuclei are central to solid-state nuclear magnetic resonance. One of the leading and best-established methods to sensitize these unreceptive species is Hartmann–Hahn cross polarization (HH-CP), a polarization transfer mechanism often executed under MAS. Herein, we explore the possibility of utilizing the 1H dipolar order created via adiabatic demagnetization in the rotating frame (ADRF), to enhance the unreceptive spins under MAS. It is found that an efficient polarization transfer via ADRF-CPMAS is not only possible but can exceed, at least in some instances involving plastic crystals, the efficiency of an optimized HH-CPMAS transfer. The experiment requires low radiofrequency nutation fields on both the 1H- and the I-spin channels, and displays unusual matching conditions that are reminiscent of the zero- and double-quantum matching conditions arising under CPMAS, albeit centered at zero frequency and demanding the simultaneous involvement of several spins. The origin of these multi-spin transfer processes is analytically derived and numerically simulated in predictions that compare well with experimental 13C and 15N results collected on model compounds at different spinning speeds. These derivations start from descriptions that depart from traditional thermodynamic arguments, and treat instead the ADRF processes in static and spinning solids on the basis of coherent evolutions. The predictions of these analytical derivations are corroborated by numerical simulations. The effects of additional factors, including chemical shift anisotropies, J-couplings, and radiofrequency inhomogeneities, are also theoretically and experimentally explored.

Analytical descriptions of (multiple-contact) cross-polarization dynamics and spin-lattice relaxation in solid alanine

In this work, several exact and approximate analytical solutions to the quantum master equation are derived using both classical and non-classical coupling models to describe the kinetics of Hartmann-Hahn cross-polarization (HHCP) and multiple-contact CP (MCCP). Moreover, the analytical solution originally obtained by Naito and McDowell [J. Chem. Phys. 84 (1986) 4181.] is shown to be incorrect and the different regimes of spin diffusion and T1ρ relaxation are characterized by the amplitude of the second stage of the HHCP dynamics and the HHCP/MCCP crossing time. The analysis of the 1H–13C HHCP and MCCP dynamics together with (Lee-Goldburg) 1H T1ρ relaxation experimental data provides a consistent picture of spin dynamics in solid alanine and explains the apparent discrepancies previously observed between T1ρ and T1 relaxation measurements. The CH and CH3 protons relax as expected via spin diffusion towards the NH3 protons but the assumption of common proton spin temperature, in which the bottleneck of relaxation is at the NH3 sites, generally valid for T1 relaxation breaks down for T1ρ relaxation. A diffusion-limited situation in which nuclear Zeeman energy is transferred to the lattice faster than can be supplied by spin diffusion is observed instead.

On the use of cross polarization in solid-state NMR: 1H spin-lock versus adiabatic demagnetization in the rotating frame

Cross polarization (CP) is a widely used solid-state nuclear magnetic resonance (NMR) technique for enhancing the polarization of dilute S spins from much larger polarization of abundant I spins such as 1H. To achieve such a polarization transfer, the I spin should either be spin-locked or be converted to the dipolar ordered state through adiabatic demagnetization in the rotating frame. In this work, we analyze the spin dynamics of the Hartmann-Hahn CP (HHCP) utilizing the 1H spin-locking, and the dipolar-order CP (DOCP) having the 1H adiabatic demagnetization. We further propose an adiabatic demagnetization CP (ADCP) where a constant radio-frequency pulse is applied on the S spin while 1H is adiabatically demagnetized. Our analyses indicate that ADCP utilizes the adiabatic passage to effectively achieve the polarization transfer from the 1H to S spins. In addition, the dipolar ordered state generated during the 1H demagnetization process could also be converted into the observable S polarization through DOCP, further enhancing the polarized signals. It is shown by both static and magic-angle-spinning (MAS) NMR experiments that ADCP has dramatically broadened the CP matching condition over the other CP schemes. Various samples have been used to demonstrate the polarization transfer efficiency of this newly proposed ADCP scheme.

Residue selective 15N CEST and CPMG experiments for studies of millisecond timescale protein dynamics

Proteins are intrinsically dynamic molecules and undergo exchanges among multiple conformations to perform biological functions. The CPMG relaxation dispersion and CEST experiments are two important solution NMR techniques for characterizing the conformational exchange processes on the millisecond timescale. Traditional pseudo 3D 15N CEST and CPMG experiments have certain limitations in their applications. For example, both experiments have low sensitivity for broadened resonances, and the process of optimizing sample conditions and experimental parameters are often time consuming. To overcome these limitations, we herein present a new set of residue selective 15N CEST and CPMG pulse sequences by employing the Hartmann-Hahn cross-polarization transfer of magnetization in both 1D and 2D schemes. Combined with frequency labeling in the indirect dimension using only a small number of increments, the pulse sequences in the 2D scheme can be applied on resonances in overlapped regions of the 1H-15N HSQC spectrum. The pulse sequences were further applied on several proteins, demonstrating their advantages over the traditional CEST and CPMG experiments under specific circumstances.

F 1 F 2-selective NMR spectroscopy.

Fourier transform NMR spectroscopy has provided unprecedented insight into the structure, interaction and dynamic motion of proteins and nucleic acids. Conventional biomolecular NMR relies on the acquisition of three-dimensional and four-dimensional (4D) data matrices to establish correlations between chemical shifts in the frequency domains F 1, F 2, F 3 and F 1, F 2, F 3, F 4 respectively. While rich in information, these datasets require a substantial amount of acquisition time, are visually highly unintuitive, require expert knowledge to process, and sample dark and bright regions of the frequency domains equally. Here, we present an alternative approach to obtain multidimensional chemical shift correlations for biomolecules. This strategy focuses on one narrow frequency range, F 1 F 2, at a time and records the resulting F 3 F 4 correlation spectrum by two-dimensional NMR. As a result, only regions of the frequency domain that contain signals in F 1 F 2 ("bright regions") are sampled. F 1 F 2 selection is achieved by Hartmann-Hahn cross-polarization using weak radio frequency fields. This approach reveals information equivalent to that of a conventional 4D experiment, while the dimensional reduction may shorten the total acquisition time and simplifies spectral processing, interpretation and comparative analysis. Potential applicability of the F 1 F 2-selective approach is illustrated by de novo assignment, structural and dynamics studies of ubiquitin and fatty-acid binding protein 4 (FABP4). Further extension of this concept may spawn new selective NMR experiments to aid studies of site-specific structural dynamics, protein-protein interactions and allosteric modulation of protein structure.

Practical considerations for investigation of protein conformational dynamics by 15N R 1ρ relaxation dispersion.

It is becoming increasingly apparent that proteins are not static entities and that their function often critically depends on accurate sampling of multiple conformational states in aqueous solution. Accordingly, the development of methods to study conformational states in proteins beyond their ground-state structure ("excited states") has crucial biophysical importance. Here we investigate experimental schemes for optimally probing chemical exchange processes in proteins on the micro- to millisecond timescale by 15N R 1ρ relaxation dispersion. The schemes use selective Hartmann-Hahn cross-polarization (CP) transfer for excitation, and derive peak integrals from 1D NMR spectra (Korzhnev et al. in J Am Chem Soc 127:713-721, 2005; Hansen et al. in J Am Chem Soc 131:3818-3819, 2009). Simulation and experiment collectively show that in such CP-based schemes care has to be taken to achieve accurate suppression of undesired off-resonance coherences, when using weak spin-lock fields. This then (i) ensures that relaxation dispersion profiles in the absence of chemical exchange are flat, and (ii) facilitates extraction of relaxation dispersion profiles in crowded regions of the spectrum. Further improvement in the quality of the experimental data is achieved by recording the free-induction decays in an interleaved manner and including a heating-compensation element. The reported considerations will particularly benefit the use of CP-based R 1ρ relaxation dispersion to analyze conformational exchange processes in larger proteins, where resonance line overlap becomes the main limiting factor.

Optimization of cross-polarization at low radiofrequency fields for sensitivity enhancement in solid-state NMR of membrane proteins reconstituted in magnetically aligned bicelles

Solid-state NMR (ssNMR) of oriented membrane proteins (MPs) is capable of providing structural and dynamic information at nearly physiological conditions. However, NMR experiments performed on oriented membrane proteins generally suffer from low sensitivity. Moreover, utilization of high-power radiofrequency (RF) irradiations for magnetization transfer may give rise to sample heating, thereby decreasing the efficiency of conventional cross-polarization schemes.Here we have optimized the recently developed repetitive cross-polarization (REP-CP) sequence (Tang et al., 2011) to further increase the magnetization transfer efficiency for membrane proteins reconstituted in magnetically aligned bicelles and compared its performance to single-contact Hartmann–Hahn cross-polarization (CP), CP-MOIST and the adiabatic transfer. It has been found that employing the REP-CP sequence at RF amplitudes of 19kHz instead of the commonly used higher RF fields (>45kHz) enhances the efficiency of REP-CP. An additional 30% signal can be obtained as compared to the previously published REP-CP, and 20% when compared to the re-optimized REP-CP at 50kHz RF fields. Moreover, the 15N signal gain of low-power REP-CP was found to be 40% over the adiabatic CP and up to 80% over CP-MOIST. Thus, the low-power REP-CP sequence surpasses all of the previous CP schemes in addition of having the tremendous advantage of reducing the RF powers by a factor of seven, thereby preserving the liquid-like bicelle sample. By contrast, in purely static (NAL crystal) and semi-rigid systems (Pf1 phage), the adiabatic CP was found to be more effective. Periodic oscillations of the intensity profile (distinct from the transient oscillations) as a function of the CP contact time and B1 RF field strengths were observed during the REP-CP optimization with the oscillations becoming more pronounced with lower RF fields. Many-spin simulations were performed to explain the oscillations and their periodicity.

Hyperpolarization of Deuterated Metabolites via Remote Cross-Polarization and Dissolution Dynamic Nuclear Polarization

In deuterated molecules such as [1-(13)C]pyruvate-d3, the nuclear spin polarization of (13)C nuclei can be enhanced by combining Hartmann-Hahn cross-polarization (CP) at low temperatures (1.2 K) with dissolution dynamic nuclear polarization (D-DNP). The polarization is transferred from remote solvent protons to the (13)C spins of interest. This allows one not only to slightly reduce build-up times but also to increase polarization levels and extend the lifetimes T1((13)C) of the enhanced (13)C polarization during and after transfer from the polarizer to the NMR or MRI system. This extends time scales over which metabolic processes and chemical reactions can be monitored.

Boosting Dissolution Dynamic Nuclear Polarization by Cross Polarization.

The efficiency of dissolution dynamic nuclear polarization can be boosted by Hartmann-Hahn cross polarization at temperatures near 1.2 K. This enables high throughput of hyperpolarized solutions with substantial gains in buildup times and polarization levels. During dissolution and transport, the (13)C nuclear spin polarization P((13)C) merely decreases from 45 to 40%.

Dissolution dynamic nuclear polarization efficiency enhanced by Hartmann–Hahn cross polarization

Dynamic nuclear polarization (DNP) can be used to increase the polarization of nuclei by utilizing the higher electron polarization. Typically, in dissolution DNP heteronuclei are directly polarized due to their longer relaxation times. We present the combination of DNP to protons with subsequent cross polarization to 13C followed by dissolution to solution state. The experiment was implemented using a modified dissolution DNP probe. For a [13C]urea sample doped with TEMPO the carbon polarization and its build-up rate could be increased by a factor of two using CP. With small losses, this gain could be transferred to the solution by dissolution.

Utilizing afterglow magnetization from cross-polarization magic-angle-spinning solid-state NMR spectroscopy to obtain simultaneous heteronuclear multidimensional spectra.

The time required for data acquisition and subsequent spectral assignment are limiting factors for determining biomolecular structure and dynamics using solid-state NMR spectroscopy. While strong magnetic dipolar couplings give rise to relatively broad spectra lines, the couplings also mediate the coherent magnetization transfer via the Hartmann-Hahn cross-polarization (HH-CP) experiment. This mechanism is used in nearly all backbone assignment experiments for carrying out polarization transfer between (1)H, (15)N, and (13)C. In this Article, we describe a general spectroscopic approach to use the residual or "afterglow" magnetization from the (15)N to (13)C selective HH-CP experiment to collect a second multidimensional heteronuclear data set. This approach allowed for the collection of two commonly used sequential assignment experiments (2D NCA and NCO or 3D NCACX and NCOCX) at the same time. Our "afterglow" technique was demonstrated with uniformly [(13)C,(15)N] and [1,3-(13)C] glycerol-labeled ubiquitin using instrumentation available on all standard solid-state NMR spectrometers configured for magic-angle-spinning. This method is compatible with several other sensitivity enhancement experiments and can be used as an isotopic filtering tool to reduce the spectral complexity and decrease the time needed for assignment.

Combining adiabatic and Hartmann–Hahn cross-polarization for sensitivity enhancement in solid state separated local field 2D-NMR experiments of partially ordered systems

In this Letter, we examine magnetization in double- and zero-quantum reservoirs of an ensemble of spin-1/2 nuclei and describe their role in determining the sensitivity of a class of separated local field NMR experiments based on Hartmann–Hahn cross-polarization. We observe that for the liquid crystal system studied, a large dilute spin-polarization, obtained initially by the use of adiabatic cross-polarization, can enhance the sensitivity of the above experiment. The signal enhancement factors, however, are found to vary and depend on the local dynamics. The experimental results have been utilized to obtain the local order-parameters of the system.

Dual Acquisition Magic‐Angle Spinning Solid‐State NMR‐Spectroscopy: Simultaneous Acquisition of Multidimensional Spectra of Biomacromolecules

Fast data collection: a general method for dual data acquisition of multidimensional magic-angle spinning solid-state NMR experiments is presented. The method uses a simultaneous Hartmann-Hahn cross-polarization from (1)H to (13)C and (15)N nuclei and exploits the long-living (15)N polarization for parallel acquisition of two multidimensional experiments.

Deuterium–carbon correlation in static oriented systems using DAPT – A windowless multiple pulse sequence

We present a new method for establishing correlation between deuterium and its attached carbon in a deuterated liquid crystal. The method is based on transfer of polarization using the DAPT pulse sequence proposed originally for two spin half nuclei, now extended to a spin-1 and a spin-1/2 nuclei. DAPT utilizes the evolution of magnetization of the spin pair under two blocks of phase shifted BLEW-12 pulses on one of the spins separated by a 90 degree pulse on the other spin. The method is easy to implement and does not need to satisfy matching conditions unlike the Hartmann–Hahn cross-polarization. Experimental results presented demonstrate the efficacy of the method.

Band-Selective1H−13C Cross-Polarization in Fast Magic Angle Spinning Solid-State NMR Spectroscopy

A magic angle spinning (MAS) NMR technique to transfer polarization from protons to a specific set of the (13)C spins is introduced for the study of biomolecular samples in the solid-state. Ultrafast (>60 kHz) MAS and low irradiation rf fields are used to achieve band-selective Hartmann-Hahn cross-polarization (CP) between the whole proton bath and carbons whose resonances are close to the (13)C-transmitter offset. When compared to conventional, broadband (1)H-(13)C CP, the band-selective experiment can be established without any loss of sensitivity when polarizing the aliphatic signals of a protein sample, and with a significant gain when polarizing carbonyls. This scheme can be used as a building block in 2D (13)C-(13)C homonuclear correlation experiments to obtain a faster and more sensitive characterization of biological solids.

Quantification of cross polarization with relaxation compensated reciprocity relation in NMR

Abstract The reciprocity relation in solid state NMR has been extended to include the effects of spin-lattice relaxation in the rotating frame. This method was successfully applied to the experiments of Hartmann–Hahn cross polarization, making the originally non-quantified NMR spectra quantitative. In addition, it provides detailed dynamics of cross polarization that is often obscured by spin-lattice relaxation in the rotating frame and by some other effects.

Thermodynamics of adiabatic cross polarization

Using a spin-temperature approach, we describe a scheme of adiabatic cross polarization, based on demagnetization/remagnetization, when the Zeeman order of abundant nuclei in the laboratory frame is first adiabatically converted into the dipolar order, and then, into the Zeeman order of rare nuclei. The scheme, implemented with two low-power frequency-sweeping pulses, is very efficient for static samples and can significantly increase polarization of rare nuclei, compared to the conventional Hartmann-Hahn cross polarization. The experimental examples are presented for a solid, liquid crystal, and small molecules in a liquid-crystalline solvent.

Operator-based triple-mode Floquet theory in solid-state NMR

Many solid-state NMR experiments exploit interference effects between time dependencies in the system Hamiltonian to design an effective time-independent Hamiltonian with the desired properties. Effective Hamiltonians can be designed such that they contain only selected parts of the full system Hamiltonian while all other parts are averaged to zero. A general theoretical description of such experiments has to accommodate several time-dependent perturbations with incommensurate frequencies. We describe an extension of the analytical operator-based Floquet description of NMR experiments to situations with three incommensurate frequencies. Experiments with three time dependencies are quite common in solid-state NMR. Examples include experiments which combine magic-angle spinning and radio-frequency irradiation on two nuclei or asynchronous multiple-pulse sequences on a single spin species. The Floquet description is general in the sense that the resulting effective Hamiltonians can be calculated without a detailed knowledge of the spin-system Hamiltonian and can be expressed fully as a function of the Fourier components of the time-dependent Hamiltonian. As a prototype experiment we treat the application of two continuous-wave (cw) radio-frequency fields under magic-angle spinning. Experiments that are included in such a description are Hartmann-Hahn cross polarization or rotary-resonance recoupling experiments with simultaneous cw decoupling.

Heteronuclear dipolar recoupling using Hartmann–Hahn cross polarization: A probe for 19F– 13C distance determination of fluorinated carbon materials

A NMR determination of the C–F bond length in fluorinated carbon materials using dipolar recoupling is described. To this end Hartmann–Hahn cross polarization with magic angle spinning (inverse cross polarization sequence) is used. A description of the corresponding 13C magnetization evolution as a function of the evolution time and its simulation for typical fluorinated samples are realized. The procedure is applied to 15 different materials having different bonding (semi-covalent or covalent) and from various carbon allotropic varieties. The distance evolves from 0.138 ± 0.002 nm for covalent bonding to 0.1445 ± 0.002 nm for semi-covalent bonding. Other parameters may affect the C–F bond length e.g. steric hindrance which leads for fluorinated fullerenes to an increase of this distance up to 0.146 ± 0.002 nm .

Heteronuclear isotropic mixing separated local field NMR spectroscopy

This paper presents a theoretical, numerical, and experimental study of a new class of separated local field (SLF) techniques. These techniques are based on the heteronuclear isotropic mixing leading to spin exchange via the local field (HIMSELF). It is shown that highly efficient and robust SLF experiments can be designed based on double channel windowless homonuclear decoupling sequences. Compared to rotating frame techniques based on Hartmann-Hahn cross polarization, the new approach is less susceptible to the frequency offset and chemical shift interaction and can be applied in the structural studies of macromolecules that are uniformly labeled with isotopes such as (13)C and (15)N. Furthermore, isotropic mixing sequences allow for transfer of any magnetization component of one nucleus to the corresponding component of its dipolar coupled partner. The performance of HIMSELF is studied by analysis of the average Hamiltonian and numerical simulation and is experimentally demonstrated on a single crystalline sample of a dipeptide and a liquid crystalline sample exhibiting motionally averaged dipolar couplings.