Heteronuclear Dipolar Couplings Research Articles

Accurate measurement of heteronuclear dipolar couplings by phase-alternating R-symmetry (PARS) sequences in magic angle spinning NMR spectroscopy.

We report a Phase-Alternating R-Symmetry (PARS) dipolar recoupling scheme for accurate measurement of heteronuclear (1)H-X (X = (13)C, (15)N, (31)P, etc.) dipolar couplings in MAS NMR experiments. It is an improvement of conventional C- and R-symmetry type DIPSHIFT experiments where, in addition to the dipolar interaction, the (1)H CSA interaction persists and thereby introduces considerable errors in the dipolar measurements. In PARS, phase-shifted RN symmetry pulse blocks applied on the (1)H spins combined with π pulses applied on the X spins at the end of each RN block efficiently suppress the effect from (1)H chemical shift anisotropy, while keeping the (1)H-X dipolar couplings intact. Another advantage over conventional DIPSHIFT experiments, which require the signal to be detected in the form of a reduced-intensity Hahn echo, is that the series of π pulses refocuses the X chemical shift and avoids the necessity of echo formation. PARS permits determination of accurate dipolar couplings in a single experiment; it is suitable for a wide range of MAS conditions including both slow and fast MAS frequencies; and it assures dipolar truncation from the remote protons. The performance of PARS is tested on two model systems, [(15)N]-N-acetyl-valine and [U-(13)C,(15)N]-N-formyl-Met-Leu-Phe tripeptide. The application of PARS for site-resolved measurement of accurate (1)H-(15)N dipolar couplings in the context of 3D experiments is presented on U-(13)C,(15)N-enriched dynein light chain protein LC8.

Quantitative Analysis of the Proximities of OH Ligands and Vanadium Sites in a Polyoxovanadate Cluster Using Frequency-Selective 1H–51V Solid-State NMR Spectroscopy

We introduce a magic-angle spinning NMR experiment to estimate specific distances in a solid material between a given site occupied by a quadrupolar nucleus and the nearby spin-1/2 nuclei. The new sequence, called DANTE-S-REDOR, consists of a frequency-selective dephasing experiment where heteronuclear dipolar couplings are reintroduced by applying a symmetry-based sequence (S-REDOR). The selectivity is achieved by applying a pulse train, such as Delays Alternating with Nutations for Tailored Excitation (DANTE), to the quadrupolar nucleus. This new method allows quantitative analysis of proximities in the 3–4 Å range of protons in OH ligands and one of the 51V sites in a complex decavanadate cluster, namely Cs4[H2V10O28]·4H2O. The high selectivity of the DANTE-S-REDOR sequence offers the possibility to investigate a wide range of materials with different quadrupolar nuclei, including polyoxometalates, oxides, zeolites, and aluminophosphates.

Multiple acquisition/multiple observation separated local field/chemical shift correlation solid-state magic angle spinning NMR spectroscopy

Multiple acquisition spectroscopy (MACSY) experiments that enable multiple free induction decays to be recorded during individual experiments are demonstrated. In particular, the experiments incorporate separated local field spectroscopy into homonuclear and heteronuclear correlation spectroscopy. The measured heteronuclear dipolar couplings are valuable in structure determination as well as in enhancing resolution by providing an additional frequency axis. In one example four different three-dimensional spectra are obtained in a single experiment, demonstrating that substantial potential saving in experimental time is available when multiple multi-dimensional spectra are required as part of solid-state NMR studies.

Phase transitions and chain dynamics of surfactants intercalated into the galleries of naturally occurring clay mineral magadiite.

We investigate conformational dynamics and phase transitions of surfactant molecules confined in the layered galleries of the organo-modified, natural polysilicate clay, magadiite. We have shown that our approach to studying this class of materials is capable of delivering detailed information on the molecular mobility of the confined molecules. From the analysis of the measured heteronuclear dipolar couplings, the orientational order parameters of the C-H bonds along the hydrocarbon chain have been determined. Three phases have been observed in the nanocomposite, characterized by distinct dynamical states of the surfactant. At room temperature, restricted mobility of the molecules led to the adoption of an essentially all-trans conformation by the chains. This behavior can be described by a model incorporating small-angle wobbling around the long molecular axes of the chains. Upon heating, dynamic transformation takes place, resulting in a rotator type solid phase where molecules in extended all-trans conformations undergo fast and unrestricted rotation about their respective symmetry axes. The second phase transition is associated with chain melting and the onset of translational dynamics and results in an essentially liquid-crystalline-like state of the organic component. The mobility of the surfactant is one of the key factors facilitating the efficient penetration of macromolecules in the process of preparing of polymer/organoclay nanocomposites. The exploration of dynamic properties of the functionalizing organic layer should provide important input into the improved design of new organic-inorganic hybrid materials.

Performance of RINEPT is amplified by dipolar couplings under ultrafast MAS conditions

The refocused insensitive nuclei enhanced by polarization transfer (RINEPT) technique is commonly used for heteronuclear polarization transfer in solution and solid-state NMR spectroscopy. Suppression of dipolar couplings, either by fast molecular motions in solution or by a combination of MAS and multiple pulse sequences in solids, enables the polarization transfer via scalar couplings. However, the presence of unsuppressed dipolar couplings could alter the functioning of RINEPT, particularly under fast/ultrafast MAS conditions. In this study, we demonstrate, through experiments on rigid solids complemented by numerical simulations, that the polarization transfer efficiency of RINEPT is dependent on the MAS frequency. In addition, we show that heteronuclear dipolar coupling is the dominant factor in the polarization transfer, which is strengthened by the presence of 1H–1H dipolar couplings. In fact, the simultaneous presence of homonuclear and heteronuclear dipolar couplings is the premise for the polarization transfer by RINEPT, whereas the scalar coupling plays an insignificant role under ultrafast MAS conditions on rigid solids. Our results additionally reveal that the polarization transfer efficiency decreases with the increasing duration of RF pulses used in the RINEPT sequence.

Dipolar Assisted Assignment Protocol (DAAP) for MAS solid-state NMR of rotationally aligned membrane proteins in phospholipid bilayers

A method for making resonance assignments in magic angle spinning solid-state NMR spectra of membrane proteins that utilizes the range of heteronuclear dipolar coupling frequencies in combination with conventional chemical shift based assignment methods is demonstrated. The Dipolar Assisted Assignment Protocol (DAAP) takes advantage of the rotational alignment of the membrane proteins in liquid crystalline phospholipid bilayers. Improved resolution is obtained by combining the magnetically inequivalent heteronuclear dipolar frequencies with isotropic chemical shift frequencies. Spectra with both dipolar and chemical shift frequency axes assist with resonance assignments. DAAP can be readily extended to three- and four-dimensional experiments and to include both backbone and side chain sites in proteins.

Homonuclear Decoupling of 1H Dipolar Interactions in Solids by means of Heteronuclear Recoupling

AbstractLine narrowing has been traditionally achieved in solid‐state 1H NMR spectroscopy by applying pulse sequences that combine multiple‐pulse operations with magic‐angle spinning (MAS), to effectively average out the dipoledipole homonuclear Hamiltonian. The present study explores a new alternative that departs from the usual concept of directly acting on the strongly coupled spins with radiofrequency pulses; instead, we seek to achieve a net homonuclear dipolar decoupling in solids by exploring the reintroduction of MAS‐averaged heteronuclear dipolar couplings between the 1H nuclei and directly bonded 13C or 15N nuclei. This recouplinganti‐recoupling (RaR) scheme thus relies on the recoupling of the dipolar interaction with heteronuclear spins, which, under fast MAS, will exceed the strength and will not commute with the homonuclear 1H1H coupling one is intending to average out. Subsequent removal (“antiRecoupling”) of these heteronuclear interactions can lead to narrowed 1H resonances, without ever pulsing on the aforementioned channel. The line‐narrowing properties of RaR are illustrated with numerical simulations and with experiments on model organic solids.

Resonance assignments of a membrane protein in phospholipid bilayers by combining multiple strategies of oriented sample solid-state NMR

Oriented sample solid-state NMR spectroscopy can be used to determine the three-dimensional structures of membrane proteins in magnetically or mechanically aligned lipid bilayers. The bottleneck for applying this technique to larger and more challenging proteins is making resonance assignments, which is conventionally accomplished through the preparation of multiple selectively isotopically labeled samples and performing an analysis of residues in regular secondary structure based on Polarity Index Slant Angle (PISA) Wheels and Dipolar Waves. Here we report the complete resonance assignment of the full-length mercury transporter, MerF, an 81-residue protein, which is challenging because of overlapping PISA Wheel patterns from its two trans-membrane helices, by using a combination of solid-state NMR techniques that improve the spectral resolution and provide correlations between residues and resonances. These techniques include experiments that take advantage of the improved resolution of the MSHOT4-Pi4/Pi pulse sequence; the transfer of resonance assignments through frequency alignment of heteronuclear dipolar couplings, or through dipolar coupling correlated isotropic chemical shift analysis; (15)N/(15)N dilute spin exchange experiments; and the use of the proton-evolved local field experiment with isotropic shift analysis to assign the irregular terminal and loop regions of the protein, which is the major "blind spot" of the PISA Wheel/Dipolar Wave method.

1H‐Observe 1H2H Dipolar Recoupling by REDOR and Rotary Resonance Recoupling

AbstractThe measurement of heteronuclear dipolar couplings by solid‐state NMR is an important tool for determining structures in solids. 1H‐observe 1H2H REDOR and rotary resonance recoupling experiments with fast magic‐angle spinning are presented. These dipolar recoupling experiments show promise for measuring heteronuclear dipolar couplings with 1H nuclei. Potential limitations of the experiments include short transverse relaxation times of the 1H magnetization and BlochSiegert effects.

Long-Lived Nuclear Spin States in Methyl Groups and Quantum-Rotor-Induced Polarization

Substances containing rapidly rotating methyl groups may exhibit long-lived states (LLSs) in solution, with relaxation times substantially longer than the conventional spin-lattice relaxation time T1. The states become long-lived through rapid internal rotation of the CH3 group, which imposes an approximate symmetry on the fluctuating nuclear spin interactions. In the case of very low CH3 rotational barriers, a hyperpolarized LLS is populated by thermal equilibration at liquid helium temperature. Following dissolution, cross-relaxation of the hyperpolarized LLS, induced by heteronuclear dipolar couplings, generates strongly enhanced antiphase NMR signals. This mechanism explains the NMR signal enhancements observed for (13)C-γ-picoline (Icker, M.; Berger, S. J. Magn. Reson. 2012, 219, 1-3).

Conformational Dynamics of Surfactant in a Mesolamellar Composite Studied by Local Field NMR Spectroscopy

Ordered mesostructured materials possess unique surface, structural, and bulk properties that lead to important practical applications. Mesostructured organic–inorganic composites are also of broad interest for fundamental studies of confinement effects and surface interaction on structural and dynamic properties of organic molecules. In the present study, solid state dipolar 13C–1H NMR spectroscopy is applied to quantitatively characterize the conformational dynamics of a surfactant in a mesolamellar composite. By applying dipolar recoupling and separated local field spectroscopy techniques, the motion of surfactant molecules was studied in a wide range of mobilities from an essentially immobilized rigid state to a highly flexible and anistropically tumbling state. From the analysis of the measured heteronuclear dipolar couplings, the orientational order parameters of C–H bonds along the surfactant chain were determined. The study shows that in surfactant bilayers in AlPO layered structure at room temper...

Heteronuclear dipolar coupling in spin-1 NQR pulsed spin locking

We investigate theoretically and experimentally the role of broadening due to heteronuclear dipolar coupling in spin-1 nuclear quadrupole resonance pulsed spin locking. We find the experimental conditions where heteronuclear dipolar coupling is refocused by a standard multipulse sequence. This experimental condition allows us to extend our previously reported ability to measure the homonuclear dipolar coupling of powder samples to include substances that have heteronuclear coupling. These results are useful for designing substance detection algorithms, and for performing sample characterization.

Resolution and measurement of heteronuclear dipolar couplings of a noncrystalline protein immobilized in a biological supramolecular assembly by proton-detected MAS solid-state NMR spectroscopy

Two-dimensional 15N chemical shift/1H chemical shift and three-dimensional 1H–15N dipolar coupling/15N chemical shift/1H chemical shift MAS solid-state NMR correlation spectra of the filamentous bacteriophage Pf1 major coat protein show single-site resolution in noncrystalline, intact-phage preparations. The high sensitivity and resolution result from 1H detection at 600MHz under 50kHz magic angle spinning using ∼0.5mg of perdeuterated and uniformly 15N-labeled protein in which the exchangeable amide sites are partially or completely back-exchanged (reprotonated). Notably, the heteronuclear 1H–15N dipolar coupling frequency dimension is shown to select among 15N resonances, which will be useful in structural studies of larger proteins where the resonances exhibit a high degree of overlap in multidimensional chemical shift correlation spectra.

Low-ε Static Probe Development for15N-1H Solid-state NMR Study of Membrane Proteins for an 800 MHz NB Magnet

H heteronuclear dipolar coupling in dilute membrane proteins oriented in hydrated anddielectrically lossy lipid environments. The system employed a 800 MHz narrow-bore magnet. A solenoid coilstrip shield was used to reduce deleterious RF sample heating by minimizing the conservative electric fieldsgenerated by the double-tuned resonator at high magnetic fields. The probe's design, construction, andperformance in solid-state NMR experiments at high magnetic fields are described here. Such high-resolutionsolid-state NMR spectroscopic analysis of static oriented samples in hydrated phospholipid bilayers or bicellescould aid the structural analysis of dilute biological membrane proteins.Key Words : Solid-state NMR probe, Low- e, Probe design, Membrane proteins, BicellesIntroductionMembrane proteins are important in various essentialsystems and regulatory processes, ranging from the com-plicated network of cellular communications to the meta-bolism and breaking down of unwanted substances in thehuman body. Hundreds of diseases involve the mis-foldingor mis-assembling of integral membrane proteins and over60% of all drug molecules target membrane proteins.Despite their importance, these protein's intrinsic structure-function relationships are not understood as their three-dimensional structures have not been fully elucidated. Thestudy of membrane proteins by current methods is hamperedby the need of a native bilayer environment, which is diffi-cult using most biophysical techniques. They are notoriouslydifficult to crystallize for X-ray crystallography and theirslow reorientation rates when combined with lipids or deter-gents hinder the use of conventional solution NMR (NuclearMagnetic Resonance) techniques. Solid-state NMR spectroscopy is a relatively new meansof investigating the structures of solid biological samples.

Detecting low-level flexibility using residual dipolar couplings: a study of the conformation of cellobiose

We have developed novel NMR methods for the measurement of heteronuclear residual dipolar couplings (RDCs) in molecules with severely overlapping NMR resonances. These and other methods enabled us to obtain 31 RDCs for α-D-cellobiose and 24 RDCs for β-D-cellobiose. The interpretation of the data in the approximation of a rigid disaccharide structure, using RDCs and interglycosidic (3)J coupling constants, yielded conformation that is very close to that determined using X-ray crystallography. However, depending on which ring was used to calculate the order parameters, the dihedral angle ψH varied up to 30° or 40°, while the φH angle was always the same. This indicates residual flexibility of the glycosidic linkage between the two monosaccharide rings and was observed for both α- and β-D-cellobiose. The RDC analysis using rigid fragments rather than a complete molecule has thus shown that the glycosidic bond of cellobiose is not completely rigid and exhibits low-level flexibility. The sources of this flexibility are discussed and evidence presented to support a hypothesis that it is associated with the ψ more than the φ angle.

Sign-sensitive determination of heteronuclear dipolar coupling to spin-1 by selective decoupling

This paper presents a theoretical and experimental study of nuclear magnetic resonance technique for sign-sensitive determination of the dipolar couplings of rare spins-1/2 to spin-1 with a strong quadrupolar interaction. The technique is based on low-power transition-selective single-quantum decoupling of spin-1 in the presence of high-power decoupling of abundant (proton) spins. Single-transition operator formalism is employed to calculate the time evolution of the density matrix in the presence of low-power irradiation of spin-1. Generally, the sign of dipolar coupling is unavailable from intrinsically symmetric shapes of dipolar-coupled spectra. Asymmetric dipolar multiplets, resulted from selective decoupling, reveal both the signs and magnitudes of the heteronuclear dipolar couplings. The approach is used to develop the experimental strategy for sign-sensitive measurements of short- and long-range heteronuclear dipolar couplings in highly ordered anisotropic samples. The technique is demonstrated for (13)C-(2)H and (13)C-(14)N spin pairs and for (13)C-(2)H(2) three-spin system in liquid crystals.

Broadband finite-pulse radio-frequency-driven recoupling (fp-RFDR) with (XY8)41 super-cycling for homo-nuclear correlations in very high magnetic fields at fast and ultra-fast MAS frequencies

We demonstrate that inter-residue 13C–13C proximities (of about 380pm) in uniformly 13C-labeled proteins can be probed by applying robust first-order recoupling during several milliseconds in single-quantum single-quantum dipolar homo-nuclear correlation (SQ–SQ D-HOMCOR) 2D experiments. We show that the intensity of medium-range homo-nuclear correlations in these experiments is enhanced using broadband first-order finite-pulse radio-frequency-driven recoupling (fp-RFDR) NMR sequence with a nested (XY8)41 super-cycling. The robustness and the efficiency of the fp-RFDR-(XY8)41 method is demonstrated at high magnetic field (21.1T) and high Magic-Angle Spinning (MAS) speeds (up to 60kHz). The introduced super-cycling, formed by combining phase inversion and a global four-quantum phase cycle, improves the robustness of fp-RFDR to (i) chemical shift anisotropy (CSA), (ii) spread in isotropic chemical shifts, (iii) rf-inhomogeneity and (iv) hetero-nuclear dipolar couplings for long recoupling times. We show that fp-RFDR-(XY8)41 is efficient sans1H decoupling, which is beneficial for temperature-sensitive biomolecules. The efficiency and the robustness of fp-RFDR-(XY8)41 is investigated by spin dynamics numerical simulations as well as solid-state NMR experiments on [U-13C]-l-histidine·HCl, a tetra-peptide (Fmoc-[U-13C,15N]-Val-[U-13C,15N]-Ala-[U-13C,15N]-Phe-Gly-t-Boc) and Al(PO3)3.

Low rf power high resolution 1H–13C–14N separated local field spectroscopy in lyotropic mesophases

Efficiency of the proton detected (encoded) 1H–13C–14N local field spectroscopy technique at low radiofrequency power is examined when applied to concentrated ionic lyotropic mesophases exhibiting heteronuclear and homonuclear dipolar couplings in kHz range. We demonstrate that highly resolved heteronuclear dipolar spectra can be obtained with limited radiofrequency power corresponding to decoupling B1 field of 20kHz and with superior spectral resolution and sensitivity provided by standard solution state probes. To maintain sufficiently large spectral window in indirect dimension, power level alternation scheme for homonuclear decoupling was introduced. In anisotropic mesophases of an ionic surfactant, pair-wise coupling constants in the three-spin system 1H–13C–14N were accurately determined. Relative signs of heteronuclear dipolar couplings to nitrogen were obtained employing frequency selective decoupling.

Improved 1H amide resonance line narrowing in oriented sample solid-state NMR of membrane proteins in phospholipid bilayers

We demonstrate 1H amide resonance line widths <300Hz in 1H/15N heteronuclear correlation (HETCOR) spectra of membrane proteins in aligned phospholipid bilayers. This represents a substantial improvement over typically observed line widths of ∼1kHz. Furthermore, in a proton detected local field (PDLF) version of the experiment that measures heteronuclear dipolar couplings, line widths <130Hz are observed. This dramatic line narrowing of 1H amide resonances enables many more individual signals to be resolved and assigned from uniformly 15N labeled membrane proteins in phospholipid bilayers under physiological conditions of temperature and pH. Finding that the decrease in line widths occurs only for membrane proteins that undergo fast rotational diffusion around the bilayer normal, but not immobile molecules, such as peptide single crystals, identifies a potential new direction for pulse sequence development that includes overall molecular dynamics in their design.

Combining dipolar–quadrupolar correlation spectroscopy with isotropic shift resolution in magic-angle-spinning 17O NMR

We explore the effect of heteronuclear dipolar recoupling on the satellite and multiple-quantum transitions of a half-integer-spin quadrupolar nucleus coupled to a single spin-12. A three-dimensional experiment is introduced that resolves different quadrupolar sites whilst allowing simultaneous extraction of the quadrupolar coupling constants, asymmetry parameters of the electric field gradient, and the isotropic shifts of the quadrupolar nucleus. The experiment also enables estimation of the heteronuclear dipolar coupling constant between the spin-12 and half-integer spin quadrupolar nucleus. The relative orientation of the dipolar tensor with respect to the quadrupolar tensor can be estimated by comparing experiments and simulations. Experimental results are shown on a sample of brucite, Mg(17OH)2, where the 1H–17O bond distance is estimated.