Rapid survey of nuclear quadrupole resonance by broadband excitation with comb modulation and dual-mode acquisition.

Nuclear Quadrupole Resonance (NQR) provides spectra carrying information as to the electric-field gradient around nuclei with a spin quantum number I > 1/2 and offers helpful clues toward characterizing the electronic structure of materials of chemical interest. A major challenge in NQR is finding hitherto unknown resonance frequencies, which can scatter over a wide range, requiring time consuming repetitive measurements with stepwise frequency increments. Here, we report on an efficient, two-step NQR protocol by bringing rapid-scan and frequency-comb together. In the first step, wideband excitation and simultaneous signal acquisition, both realized by a non-adiabatic, frequency-swept hyperbolic secant (HS) pulse with comb modulation, offers a clue for the existence/absence of the resonance within the frequency region under investigation. When and only when the sign of the resonance has been detected, the second step is implemented to compensate the limited detection bandwidth of the first and to unambiguously determine the NQR frequency. We also study the spin dynamics under the comb-modulated HS pulse by numerical simulations, and experimentally demonstrate the feasibility of the proposed scheme, which is referred to as RApid-Scan with GApped excitation with Dual-mode Operation (RASGADO) NQR.

Terminal and bridging fluorine ligands in TiF4 as studied by 19F NMR in solids.

To examine bonding nature of fluorine ligands in a metal coordinated system, 19F high-resolution solid-state NMR has been applied to TiF4, which bears both bridging and terminal fluorines. Observed 12 isotropic signals are assigned to 12 crystallographically different fluorines (6 terminal and 6 bridging fluorines) in TiF4 by referring to the calculated isotropic shifts using density functional theory (DFT). The isotropic chemical shift (δiso) for terminal F (FT) appears at high frequency (420-480 ppm from δ(CCl3F) = 0 ppm) with large shielding anisotropy Δσ ∼ 850 ppm. Whereas the δiso and Δσ values for bridging F (FB) are moderate; δiso ∼ 0-25 ppm and Δσ ∼ 250 ppm. The origin of the observed high-frequency shift for FT is ascribed to the second-order paramagnetic shift with increased covalency, shorter Ti-F bonds, and smaller energy difference between the occupied and vacant orbitals. Examination of the orientation of the shielding tensor relative to the molecular structure shows that the most deshielded component of the shielding tensor is oriented along the Ti-F bond. The characteristic orientation is consistent with a Ti-F σ bond formed by dYZ of Ti and pz of F. Further, we show that the selectively observed spinning sideband patterns and the theoretical patterns with the calculated Δσ and η (shielding asymmetry) values are not consistent with each other for FB, indicating deficiency of the present DFT calculation in evaluating Δσ.

Solid-State Nuclear Magnetic Resonance Analysis Reveals a Possible Calcium Binding Site of Pradimicin A.

Pradimicin A (PRM-A) is a unique natural product that recognizes d-mannopyranoside (Man) in the presence of Ca2+ ion. Although the Man binding geometry of PRM-A is largely understood, the molecular basis of Man recognition has yet to be established because of the lack of information regarding Ca2+ binding geometry. In this work, to examine the Ca2+ binding site of PRM-A, we performed a solid-state nuclear magnetic resonance experiment using 111Cd2+ as a surrogate probe for Ca2+. Evaluation of 13C-111Cd distances in the [PRM-A/111Cd2+] complexes by rotational-echo double resonance (REDOR) and 111Cd frequency selective REDOR (FSR) revealed that PRM-A binds 111Cd2+ at the anthraquinone moiety, which contradicts the previous hypothesis of the alanine moiety being the Ca2+ and Cd2+ binding sites of PRM-A. The distances between Cd2+ and the carbon atoms at the binding site of PRM-A were found to be 3.5 ± 0.2 Å. Importantly, Man binding was shown not to alter the distances, indicating that [PRM-A/Ca2+] and [PRM-A/Ca2+/Man] complexes have similar Ca2+ binding geometries. This study provides an important clue to understanding the molecular basis of Man recognition of PRM-A.

Rotational resonance for a heteronuclear spin pair under magic-angle spinning in solid-state NMR.

Rotational resonance (R2) is one of the widely applied techniques in solid-state NMR for recoupling a homonuclear dipolar interaction under magic-angle spinning (MAS). R2 occurs as the result of interference between the difference of the chemical shifts and MAS. In this work, we extend R2 to a heteronuclear dipolar interaction in the interaction frame of RF irradiation. Based on the average Hamiltonian theory, we show that the recoupling of the heteronuclear dipolar (I-S) interaction occurs at the recoupling conditions written as ΩI'±ΩS'=kωr (k=0,±1,±2), where ΩX' is the RF offset for spin-X (X = I or S) scaled by RF irradiation. The new recoupling sequence for a heteronuclear spin pair is referred to as offset-driven cross polarization along the z axis (OD-CPZ). With the robustness for RF inhomogeneity and ten recoupling conditions to choose, OD-CPZ can be a useful frequency-selective cross polarization method. Experiments and numerical simulations are shown with the results of the theoretical analysis.

Determination of nuclear quadrupolar parameters using singularities in field-swept NMR patterns.

We propose a simple data-analysis scheme to determine the coupling constant and the asymmetry parameter of nuclear quadrupolar interactions in field-swept nuclear magnetic resonance (NMR) for static powder samples. This approach correlates the quadrupolar parameters to the positions of the singularities, which can readily be found out as sharp peaks in the field-swept pattern. Moreover, the parameters can be determined without quantitative acquisition and elaborate calculation of the overall profile of the pattern. Since both experimental and computational efforts are significantly reduced, the approach presented in this work will enhance the power of the field-swept NMR for yet unexplored quadrupolar nuclei. We demonstrate this approach in 33S in α-S8 and 35Cl in chloranil. The accuracy of the obtained quadrupolar parameters is also discussed.

Comparison among Magnus/Floquet/Fer expansion schemes in solid-state NMR.

We here revisit expansion schemes used in nuclear magnetic resonance (NMR) for the calculation of effective Hamiltonians and propagators, namely, Magnus, Floquet, and Fer expansions. While all the expansion schemes are powerful methods there are subtle differences among them. To understand the differences, we performed explicit calculation for heteronuclear dipolar decoupling, cross-polarization, and rotary-resonance experiments in solid-state NMR. As the propagator from the Fer expansion takes the form of a product of sub-propagators, it enables us to appreciate effects of time-evolution under Hamiltonians with different orders separately. While 0th-order average Hamiltonian is the same for the three expansion schemes with the three cases examined, there is a case that the 2nd-order term for the Magnus/Floquet expansion is different from that obtained with the Fer expansion. The difference arises due to the separation of the 0th-order term in the Fer expansion. The separation enables us to appreciate time-evolution under the 0th-order average Hamiltonian, however, for that purpose, we use a so-called left-running Fer expansion. Comparison between the left-running Fer expansion and the Magnus expansion indicates that the sign of the odd orders in Magnus may better be reversed if one would like to consider its effect in order.

Proton decoupling and recoupling under double-nutation irradiation in solid-state NMR.

The effect of (1)H decoupling in magic-angle spinning solid-state NMR is studied under radiofrequency irradiation causing simultaneous nutations around a pair of orthogonal axes. Double-nutation with an arbitrary pair of nutation frequencies is implemented through modulation of the amplitude, phase, and frequency of the transmitting pulses. Similarity and difference of double-nutation decoupling and two-pulse phase-modulation decoupling schemes [A. E. Bennett, C. M. Rienstra, M. Auger, K. V. Lakshmi, and R. G. Griffin, J. Chem. Phys. 103, 6951-6958 (1995) and I. Scholz, P. Hodgkinson, B. H. Meier, and M. Ernst, J. Chem. Phys. 130, 114510 (2009)] are discussed. The structure of recoupling bands caused by interference of the (1)H spin nutation with sample spinning is studied by both experiments and numerical simulations.

Mannose-binding geometry of pradimicin A.

Pradimicins (PRMs) and benanomicins are the only family of non-peptidic natural products with lectin-like properties, that is, they recognize D-mannopyranoside (Man) in the presence of Ca(2+) ions. Coupled with their unique Man binding ability, they exhibit antifungal and anti-HIV activities through binding to Man-containing glycans of pathogens. Notwithstanding the great potential of PRMs as the lectin mimics and therapeutic leads, their molecular basis of Man recognition has yet to be established. Their aggregate-forming propensity has impeded conventional interaction analysis in solution, and the analytical difficulty is exacerbated by the existence of two Man binding sites in PRMs. In this work, we investigated the geometry of the primary Man binding of PRM-A, an original member of PRMs, by the recently developed analytical strategy using the solid aggregate composed of the 1:1 complex of PRM-A and Man. Evaluation of intermolecular distances by solid-state NMR spectroscopy revealed that the C2-C4 region of Man is in close contact with the primary binding site of PRM-A, while the C1 and C6 positions of Man are relatively distant. The binding geometry was further validated by co-precipitation experiments using deoxy-Man derivatives, leading to the proposal that PRM-A binds not only to terminal Man residues at the non-reducing end of glycans, but also to internal 6-substituted Man residues. The present study provides new insights into the molecular basis of Man recognition and glycan specificity of PRM-A.

A statistical approach for analyzing the development of 1H multiple-quantum coherence in solids.

A novel statistical approach for analyzing (1)H multiple-quantum (MQ) spin dynamics in so-called spin-counting solid-state NMR experiments is presented. The statistical approach is based on the percolation theory with Monte Carlo methods and is examined by applying it to the experimental results of three solid samples having unique hydrogen arrangement for 1-3 dimensions: the n-alkane/d-urea inclusion complex as a one-dimensional (1D) system, whose (1)H nuclei align approximately in 1D, and magnesium hydroxide and adamantane as a two-dimensional (2D) and a three-dimensional (3D) system, respectively. Four lattice models, linear, honeycomb, square and cubic, are used to represent the (1)H arrangement of the three samples. It is shown that the MQ dynamics in adamantane is consistent with that calculated using the cubic lattice and that in Mg(OH)2 with that calculated using the honeycomb and the square lattices. For n-C20H42/d-urea, these 4 lattice models fail to express its result. It is shown that a more realistic model representing the (1)H arrangement of n-C20H42/d-urea can describe the result. The present approach can thus be used to determine (1)H arrangement in solids.

Exploring various modulation-sideband recoupling conditions of SHA+ sequence at fast MAS.

We explore modulation-sideband recoupling conditions of the (13)C-(13)C Second-order Hamiltonian among Analogous nuclei plus pulse sequence (SHA+), and found that this sequence can be used in two different recoupling regimes. The first regime, νR>Δνiso(max), is recommended for broad-band recoupling to avoid any rotational resonance broadening. In this regime, the spinning speed should be only slightly larger than Δνiso(max), to obtain the best transfer efficiency. The second regime, νR<Δνiso(max), can be used to observe long-range constraints with lower spinning speed, which increases the transfer efficiency, and may allow using bigger rotors to increase the S/N ratio.

Solid-state NMR analysis of the ß-strand orientation of the protofibrils of amyloid ß-protein.

Alzheimer's disease (AD) is caused by abnormal deposition (fibrillation) of a 42-residue amyloid ß-protein (Aß42) in the brain. During the process of fibrillation, the Aß42 takes the form of protofibrils with strong neurotoxicity, and is thus believed to play a crucial role in the pathogenesis of AD. To elucidate the supramolecular structure of the Aß42 protofibrils, the intermolecular proximity of the Ala-21 residues in the Aß42 protofibrils was analyzed by (13)C-(13)C rotational resonance experiments in the solid state. Unlike the Aß42 fibrils, an intermolecular (13)C-(13)C correlation was not found in the Aß42 protofibrils. This result suggests that the ß-strands of the Aß42 protofibrils are not in an in-register parallel orientation. Aß42 monomers would assemble to form protofibrils with the ß-strand conformation, then transform into fibrils by forming intermolecular parallel ß-sheets.

Very-long-distance correlations in proteins revealed by solid-state NMR spectroscopy.

I still see you: a new pulse sequence, SHA+, little sensitive to dipolar truncation, allows direct or relayed polarization transfer between (13)C atoms, distant by 3.5-9.6 Å, in amyloid fibrils. SHA+ can also be used in a broadband way with the weak rf-condition of ν(1)/ν(R)≈0.2-0.3 which permits the investigation of temperature-sensitive biological systems.

Solid-state NMR analysis of calcium and d-mannose binding of BMY-28864, a water-soluble analogue of pradimicin A.

Pradimicin A (PRM-A) is a unique antibiotic with a lectin-like ability to recognize d-mannopyranosides (Man) in the presence of Ca(2+) ion. BMY-28864 (1) is a water-soluble analogue of PRM-A, which has been extensively used for studies on the mode of Man recognition and antifungal action of pradimicins. Although it has been assumed that PRM-A and 1 bind Man in a similar fashion, direct experimental evidence has yet to be provided. In this report, we compared Ca(2+) and Man binding of 1 with that of PRM-A through two solid-state NMR experiments. The solid-state (113)Cd NMR analysis using (113)Cd(2+) ion as a surrogate for Ca(2+) ion suggested the similarity in Ca(2+) coordination of PRM-A and 1. The dipolar assisted rotational resonance (DARR) analysis using (13)C-labeled 1 clearly showed that 1 as well as PRM-A binds Man near its carboxyl group. These results collectively indicate that the mode of binding of Ca(2+) ion and Man is nearly identical between PRM-A and 1.

Homo- and heteronuclear two-dimensional covariance solid-state NMR spectroscopy with a dual-receiver system.

Two-dimensional (2D) covariance NMR spectroscopy, which has originally been established to extract homonuclear correlations (HOMCOR), is extended to include heteronuclear correlations (HETCOR). In a (13)C/(15)N 2D chemical shift correlation experiment, (13)C and (15)N signals of a polycrystalline sample of (13)C, (15)N-labeled amino acid are acquired simultaneously using a dual-receiver NMR system. The data sets are rearranged for the covariance data processing, and the (13)C-(15)N heteronuclear correlations are obtained together with the (13)C-(13)C and (15)N-(15)N homonuclear correlations. The present approach retains the favorable feature of the original covariance HOMCOR that the spectral resolution along the indirect dimension is given by that of the detection dimension. As a result, much fewer amounts of data are required to obtain a well-resolved 2D spectrum compared to the case of the conventional 2D Fourier-Transformation (FT) scheme. Hence, one can significantly save the experimental time, or enhance the sensitivity by increasing the number of signal averaging within a given measurement time.

Mapping of the primary mannose binding site of pradimicin A.

Pradimicin A (PRM-A) is an actinomycete-derived antibiotic with the lectin-like property of being able to recognize D-mannopyranoside (Man) in the presence of Ca(2+) ion. PRM-A and its derivatives have been attracting a great deal of attention as the only family of natural carbohydrate receptors with nonpeptidic skeleton and, more recently, as conceptually novel drug candidates for human immunodeficiency virus (HIV). Despite its scientific interest and potential therapeutic importance, understanding how PRM-A recognizes Man has been severely limited. Conventional interaction analysis of PRM-A with Man in solution has been frustrated by aggregation of PRM-A and the three-component equilibrium consisting of the [PRM-A(2)/Ca(2+)], [PRM-A(2)/Ca(2+)/Man(2)], [PRM-A(2)/Ca(2+)/Man(4)] complexes, and their mixed oligomers. In this Article, we demonstrate the interaction analysis of PRM-A with methyl α-D-mannopyranoside (Man-OMe) in the solid state, which benefits from aggregate-forming propensity of PRM-A and eliminates the problem associated with the complicated equilibrium in solution. Isothermal titration calorimetry (ITC) analysis and coprecipitation experiments revealed that the primary Man binding of PRM-A is markedly tighter than the secondary one, leading to preparation of the solid aggregate solely composed of the [PRM-A(2)/Ca(2+)/Man-OMe(2)] complex. The simple 1:1 complexes of biosynthetically (13)C-enriched PRM-As and [(13)C(6)]Man-OMe facilitated the analysis of the primary Man binding of PRM-A by two-dimensional dipolar-assisted rotational resonance (2D-DARR), which clearly identified that the cavity consisted of D-alanine moiety and ABC rings of PRM-A is the Man binding site. Interestingly, the proposed Man binding site of PRM-A seems to resemble the typical architecture of artificial carbohydrate receptors.

Sensitive (13)C- (13)C correlation spectra of amyloid fibrils at very high spinning frequencies and magnetic fields.

Sensitive 2D solid-state (13)C-(13)C correlation spectra of amyloid ß fibrils have been recorded at very fast spinning frequencies and very high magnetic fields. It is demonstrated that PARIS-xy recoupling using moderate rf amplitudes can provide structural information by promoting efficient magnetization transfer even under such challenging experimental conditions. Furthermore, it has been shown both experimentally and by numerical simulations that the method is not very sensitive to dipolar truncation effects and can reveal direct transfer across distances of about 3.5-4 Å.

Solid-state NMR analysis of interaction sites of curcumin and 42-residue amyloid ß-protein fibrils.

Aggregation of 42-residue amyloid ß-protein (Aß42) plays a pivotal role in the etiology of Alzheimer's disease (AD). Curcumin, the yellow pigment in the rhizome of turmeric, attracts considerable attention as a food component potentially preventing the pathogenesis of AD. This is because curcumin not only inhibits the aggregation of Aß42 but also binds to its aggregates (fibrils), resulting in disaggregation. However, the mechanism of interaction between curcumin and the Aß42 fibrils remains unclear. In this study, we analyzed the binding mode of curcumin to the Aß42 fibrils by solid-state NMR using dipolar-assisted rotational resonance (DARR). To improve the quality of 2D spectra, 2D DARR data were processed with the covariance NMR method, which enabled us to detect weak cross peaks between carbons of curcumin and those of the Aß42 fibrils. The observed (13)C-(13)C cross peaks indicated that curcumin interacts with the 12th and 17-21st residues included in the ß-sheet structure in the Aß42 fibrils. Interestingly, aromatic carbons adjacent to the methoxy and/or hydroxy groups of curcumin showed clear cross peaks with the Aß42 fibrils. This suggested that these functional groups of curcumin play an important role in its interaction with the Aß42 fibrils.

(14)N quadrupolar coupling of amide nitrogen and Peptide secondary structure as studied by solid-state NMR spectroscopy.

To establish a relationship between the secondary structure of a peptide and the quadrupolar coupling of its amide (14)N, we examined (14)N quadrupolar couplings for eight different polypeptide samples, each of whose secondary structure (alpha-helix or beta-sheet) is known. The (14)N quadrupolar coupling is estimated from indirect observation of a (14)N overtone resonance under magic-angle spinning. From the observed indirect (14)N overtone spectra and calculated (14)N quadrupolar couplings for model molecules by using ab initio calculation (Gaussian03), it is shown that the quadrupolar coupling for the alpha-helix is larger than that for the beta-sheet by a few 100 kHz irrespective of the kind of amino acid residues examined (Ala, Val, Leu).

Phase covariance in NMR signal.

To examine noisy nuclear-magnetic resonance (NMR) spectra, we have developed a novel signal analysis method based on phase correlation between the NMR signal and the excitation pulse. The new phase-covariance analysis is compatible with the conventional signal accumulation, and successful de-noising is demonstrated.

Identification of physiological and toxic conformations in Abeta42 aggregates.

Aggregation of the 42-residue amyloid beta-protein (Abeta42) plays a crucial role in the pathogenesis of Alzheimer's disease (AD). Despite numerous structural studies on Abeta aggregates, the relationship between tertiary structure and toxicity remains unclear. Our proline scanning and solid-state NMR studies suggested that aggregates both of wild-type Abeta42 and of E22K-Abeta42 (one of the mutants related to cerebral amyloid angiopathy) contain two conformers: a major one with a turn at positions 25 and 26, and a minor one with a turn at positions 22 and 23. To identify the toxic conformer, the derivative Abeta42-lactam(22K-23E), in which the side chains at positions 22 and 23 were covalently linked, was synthesized as a minor conformer surrogate, along with Abeta42-lactam(25K-26E) as a major conformer surrogate. The Abeta42-lactam(22K-23E) showed stronger aggregation, neurotoxicity, radical generation, and oligomerization than wild-type Abeta42, whereas in Abeta42-lactam(25K-26E) were weak. The transition from the physiological conformation with a turn at positions 25 and 26 to the toxic conformation with a turn at positions 22 and 23 might be a key event in the pathogenesis of AD.