New methods for calibration of the RF field strength for indirectly observed nuclei

Two novel pulse sequences, CALIS-1 and CALIS-2, for accurate calibration of the RF field strength for an indirectly observed spin are introduced. CALIS-2 is intended for calibration of e.g., (13)C or (15)N pulses on natural abundance samples whilst CALIS-1 is recommended primarily for enriched samples. Both experiments can be performed without prior knowledge or guess of the RF field strength and no delays in the pulse sequences are critically dependent on coupling constants.     

Sensitivity and resolution enhancement in solid-state NMR spectroscopy of bicelles

Magnetically aligned bicelles are becoming attractive model membranes to investigate the structure, dynamics, geometry, and interaction of membrane-associated peptides and proteins using solution- and solid-state NMR experiments. Recent studies have shown that bicelles are more suitable than mechanically aligned bilayers for multidimensional solid-state NMR experiments. In this work, we describe experimental aspects of the natural abundance (13)C and (14)N NMR spectroscopy of DMPC/DHPC bicelles. In particular, approaches to enhance the sensitivity and resolution and to quantify radio-frequency heating effects are presented. Sensitivity of (13)C detection using single pulse excitation, conventional cross-polarization (CP), ramp-CP, and NOE techniques are compared. Our results suggest that the proton decoupling efficiency of the FLOPSY pulse sequence is better than that of continuous wave decoupling, TPPM, SPINAL, and WALTZ sequences. A simple method of monitoring the water proton chemical shift is demonstrated for the measurement of sample temperature and calibration of the radio-frequency-induced heating in the sample. The possibility of using (14)N experiments on bicelles is also discussed.     

Sensitivity enhancement of a two-dimensional experiment for the measurement of heteronuclear long-range coupling constants, by a new scheme of coherence selection by gradients

In this work we present a new pulse sequence for the measurement of long-range heteronuclear coupling constants in which the optimization of coherence selection by pulsed field gradients offers a net increase in sensitivity. This type of experiments is extremely valuable for conformational studies of molecules in natural abundance and in this context the use of gradients is essential for an efficient suppression of (12)C bound proton signals. A comparative analysis of the different gradient schemes available is presented with a conclusive elucidation of the relative sensitivities. Our gradient scheme could be advantageous as a building block for other related experiments.     

The chemical shift interaction under weak radio frequency pulses

The chemical shift anisotropy of a nuclear spin system in a strong magnetic field can be comparable to the strength of the rf pulse (weak pulse condition). In this case, the commonly used assumption that the chemical shift interaction Hamiltonian in the duration of a rf pulse can be neglected is no longer effective. The rf response characteristics of a spin-1/2 system under a weak pulse condition is studied in detail. The relationships between the distortion of the chemical shift powder spectrum and the relative rf field amplitude under various conditions are given. The suppression of sidebands of a MAS spectrum is analysed as an example of solid multiple pulse experiments. The experimental results are in good agreement with computer simulations.     

13C natural abundance S3E and S3CT experiments for measurement of J coupling constants between 13Calpha or 1Halpha and other protons in a protein

It is demonstrated that the spin-state-selective pulse sequence elements, S3E and S3CT, previously introduced for measurement of J coupling constants in 15N-labeled proteins can be applied for work with peptides and proteins with 13C at the natural abundance level. In addition, a method is described for suppression of crosstalk caused by passive spin flips and pulse imperfections, which otherwise results in systematically underestimated J coupling constants and thereby inaccurate structural constraints. This method is also applicable for crosstalk suppression in applications of S3E and S3CT to 13C- or 15N-labeled samples. Experimental confirmation is obtained using a 10 mM BPTI sample focusing on 13C in the alpha position. The measured J coupling constants include 3J(HN-Halpha) and 3J(Halpha-Hbeta) related to the phi and chi1 angles, respectively.     

Coherence selection in double CP MAS NMR spectroscopy

Applications of double cross-polarization (CP) magic-angle spinning (MAS) NMR spectroscopy, via (1)H/(15)N and then (15)N/(13)C coherence transfers, for (13)C coherence selection are demonstrated on a (15)N/(13)C-labeled N-acetyl-glucosamine (GlcNAc) compound. The (15)N/(13)C coherence transfer is very sensitive to the settings of the experimental parameters. To resolve explicitly these parameter dependences, we have systematically monitored the (13)C{(15)N/(1)H} 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 (13)C effective rf field is larger than that of the (15)N 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.     

Adjusting Conditions for High-resolution Pulsed NMR in Solids

Adjusting conditions of pulse sequences for high-resolution nmr in solids are demonstrated by the example of the WHH4 cycle. For this pulse sequence the well-known adjusting condition [1] occurs as limiting case at sufficiently weak dipolar interaction. The relations between the pulse rotation angle and interval time proved to be dependent on the dipolar interaction. Therefore it is impossible to suppress the dipolar interaction in ordinary solids completely. If the strength of the dipolar interaction varies in a range depending on the allowable inhomogeneity of the rf field a spectrometer for high-resolution nmr in solids can be adjusted in an almost optimal way.     

Selective averaging for high-resolution solid-state NMR spectroscopy of aligned samples

Solid-state NMR experiments benefit from being performed at high fields, and this is essential in order to obtain spectra with the resolution and sensitivity required for applications to protein structure determination in aligned samples. Since the amount of rf power that can be applied is limited, especially for aqueous protein samples, the most important pulse sequences suffer from bandwidth limitations resulting from the same spread in chemical shift frequencies that aids resolution. SAMPI4 is a pulse sequence that addresses these limitations. It yields separated local field spectra with narrower and more uniform linewidths over the entire spectrum than the currently used PISEMA and SAMMY experiments. In addition, it is much easier to set up on commercial spectrometers and can be incorporated as a building block into other multidimensional pulse sequences. This is illustrated with a two-dimensional HETCOR experiment, where it is crucial to transfer polarization from the amide protons to their directly bonded nitrogens over a wide range of chemical shift frequencies. A quantum-mechanical treatment of the spin Hamiltonians under high-power rf pulses is presented which gives the scaling factor for SAMPI4 as well as the durations of the rf pulses to achieve optimal decoupling.     

Optimal control based NCO and NCA experiments for spectral assignment in biological solid-state NMR spectroscopy

We present novel pulse sequences for magic-angle-spinning solid-state NMR structural studies of (13)C,(15)N-isotope labeled proteins. The pulse sequences have been designed numerically using optimal control procedures and demonstrate superior performance relative to previous methods with respect to sensitivity, robustness to instrumental errors, and band-selective excitation profiles for typical biological solid-state NMR applications. Our study addresses specifically (15)N to (13)C coherence transfers being important elements in spectral assignment protocols for solid-state NMR structural characterization of uniformly (13)C,(15)N-labeled proteins. The pulse sequences are analyzed in detail and their robustness towards spin system and external experimental parameters are illustrated numerically for typical (15)N-(13)C spin systems under high-field solid-state NMR conditions. Experimentally the methods are demonstrated by 1D (15)N-->(13)C coherence transfer experiments, as well as 2D and 3D (15)N,(13)C and (15)N,(13)C,(13)C chemical shift correlation experiments on uniformly (13)C,(15)N-labeled ubiquitin.     

Supercycled symmetry-based double-quantum dipolar recoupling of quadrupolar spins in MAS NMR: I. Theory

Using average Hamiltonian (AH) theory, we analyze recently introduced homonuclear dipolar recoupling pulse sequences for exciting central-transition double-quantum coherences (2QC) between half-integer spin quadrupolar nuclei undergoing magic-angle-spinning. Several previously observed differences among the recoupling schemes concerning their compensation to resonance offsets and radio-frequency (rf) inhomogeneity may qualitatively be rationalized by an AH analysis up to third perturbation order, despite its omission of first-order quadrupolar interactions. General aspects of the engineering of 2Q-recoupling pulse sequences applicable to half-integer spins are discussed, emphasizing the improvements offered from a diversity of supercycles providing enhanced suppression of undesirable AH cross-terms between resonance offsets and rf amplitude errors.     

Theoretical studies of the effect of the dipolar field in multiple spin-echo sequences with refocusing pulses of finite duration

It has been observed recently that the finite duration of refocusing rf pulses in a multiecho acquisition of the signal formed under the influence of the dipolar field leads to significant signal attenuation [S. Kennedy, Z. Chen, C.K. Wong, E.W.-C. Kwok, J. Zhong, Investigation of multiple-echo spin-echo signal acquisition under distant dipole-dipole interactions, Proc. Int. Soc. Magn. Reson. Med. 13 (2005) 2288]. Hereto, we quantify the phenomenon by evaluating analytically the influences of both the distant dipolar field (DDF) and transverse relaxation T2 on the magnetization in a multiecho pulse sequence based on correlation spectroscopy revamped by asymmetric z-gradient echo detection (CRAZED). Analytic expressions for the magnetization were obtained, which demonstrate explicitly the origin of rephased signal in the presence of the finite pi pulses in the multiecho train. The expressions also explain the effects of the DDF and T2 during the refocusing pulses on the signal strength, and show the substantial signal dependence on the phase of the rf pulses. We show that when the DDF effect during the pulse is canceled, the signal rises primarily during the free evolution time in the acquisition period. This elucidates the signal attenuation when the rf pulses cover a significant proportion of time in the sequence. In addition, we performed an optimization on the number of refocusing pulses that maximizes the total acquired signal using parameters for water, brain white matter, and muscle. We found that maximal signal-to-noise ratio is obtained when the pulse duration approximately equals the free evolution time in the samples with a wide range of T2.     

Broadband dipolar recoupling in rotating solids: a numerical comparison of some pulse schemes

A numerical comparison of the dipolar recoupling performance of several previously published homonuclear recoupling schemes under magic angle-spinning conditions is presented. Emphasis is put on the recoupled polarization transfer in a two-spin system where the efficiency is studied as a function of resonance offsets in the presence and absence of chemical-shielding anisotropies. In addition, the effect of the rf field strength is investigated. Powder pattern line shapes are shown in the on-resonance case that reveal the distribution of dipolar couplings for each recoupling scheme. These results are compared to data computed with a purposely misset rf field strength to estimate the pulse scheme sensitivity to rf-inhomogeneity and experimental missettings.     

Spin-state-selective excitation in selective 1D inverse NMR experiments

A general and very simple strategy for achieving clean spin-state-selective excitation with full sensitivity in carbon-selective gradient-enhanced 1D HMQC and HSQC pulse schemes is presented. The incorporation of an additional hard 90 degrees (13)C pulse applied along a specific orthogonal axis just prior to acquisition into the conventional sequences allows us to select a simultaneous coherence transfer pathway which usually is not detected. The superimposition of this resulting antiphase magnetization to the conventional in-phase magnetization gives the exclusive excitation of the directly attached proton showing only the alpha or beta spin state of the passive (13)C nucleus. The propagation of this particular spin state to other protons can be accomplished by adding any homonuclear mixing process just after this supplementary pulse. Such an approach affords a suite of powerful selective 1D (13)C-edited NMR experiments which are helpful for resonance assignment purposes in overcrowded proton spin systems and also for the accurate determination of the magnitude and sign of long-range proton-carbon coupling constants in CH spin sytems for samples at natural abundance. Such measurements are performed by measuring the relative displacement of relayed signals in the corresponding alpha and beta 1D subspectra.     

Applications of DNP-NMR for the measurement of heteronuclear T1 relaxation times

Measurement of heteronuclear spin-lattice relaxation times is hampered by both low natural abundance and low detection sensitivity. Combined with typically long relaxation times, this results in extended acquisition times which often renders the experiment impractical. Recently a variant of dynamic nuclear polarisation has been demonstrated in which enhanced nuclear spin polarisation, generated in the cryo-solid state, is transferred to the liquid state for detection. Combining this approach with small flip angle pulse trains, similar to the FLASH-T(1) imaging sequence, allows the rapid determination of spin-lattice relaxation times. In this paper we explore this method and its application to the measurement of T(1) for both carbon-13 and nitrogen-15 at natural abundance. The effects of RF inhomogeneity and the influence of proton decoupling in the context of this experiment are also investigated.     

Satellite transitions in natural abundance solid-state (33)S MAS NMR of alums--sign change with zero-crossing of C(Q) in a variable temperature study

Experiences obtained from recent improvements in the performance of solid-state (14)N MAS NMR spectroscopy have been used in a natural abundance (33)S MAS NMR investigation of the satellite transitions for this interesting spin I=3/2 isotope. This study reports the first observation of manifolds of spinning sidebands for these transitions in (33)S MAS NMR as observed for the two alums XAl(SO(4))(2) x 12H(2)O with X=NH(4) and K. For the NH(4)-alum a variable temperature (33)S MAS NMR study, employing the satellite transitions, shows that the (33)S quadrupole coupling constant (C(Q)) exhibits a linear temperature dependence (in the range -35 degrees C to 70 degrees C) with a temperature gradient of 3.1 kHz/ degrees C and undergoes a sign change with zero-crossing for C(Q) at 4 degrees C (277 K). For the isostructural K-alum a quite similar increase in the magnitude of C(Q) with increasing temperature is observed, and with a temperature gradient of 2.3 kHz/ degrees C. Finally, for optimization purposes, a study on the effect of the applied pulse widths at constant rf field strength on the intensity and variation in second-order quadrupolar lineshape for the central (1/2<-->-1/2) transition of the K-alum has been performed.     

Optimum excitation of “enhanced” central transition populations

Central transition (CT) sensitivity enhancement schemes that transfer polarization from satellites to the CT through selective saturation or inversion of neighboring satellite transitions have provided a welcome improvement for magic-angle spinning spectra of half-integer quadrupole nuclei. While many researchers have investigated and developed different methods of creating enhanced CT populations, here we investigate the conversion of these enhanced CT populations into observable CT coherence. We show a somewhat unexpected result that a conversion pulse length optimized for maximum sensitivity on equilibrium populations may not be optimum for an enhanced (non-equilibrium) polarization. Furthermore, CT enhancements can be lost if excessive rf field strength is used to convert this enhanced polarization into CT coherence. While a maximally enhanced CT signal is expected when using a perfectly selective CT conversion pulse, we have found that significant sensitivity loss can occur when using surprisingly low rf field strengths, even for sites with relatively large quadrupole coupling constants. We have systematically investigated these issues, and present some general guidelines and expectations when optimizing the conversion of enhanced (non-equilibrium) CT populations into observable CT coherence.     

General implementation of the ERETIC method for pulsed field gradient probe heads

A capacitive coupling between a secondary radiofrequency (rf) channel and the gradient coil of a standard commercially available high resolution NMR spectrometer and probe head is described and used to introduce a low level exponentially damped rf signal near the frequency of the primary rf channel to serve as an external concentration standard, in analogy to the so-called ERETIC™ method. The stability of this inexpensive and simple to implement method, here referred to as the Pulse Into the Gradient (PIG) approach, is superb over a 14-h period and both gradient tailored water suppression and one-dimensional imaging applications are provided. Since the low level signal is introduced via the pulsed field gradient coil, the coupling is identical to that for a free induction signal and thus the method proves to be immune (within 5%) to sample ionic strength effects up to the 2 M NaCl solutions explored here.     

Heteronuclear Double-Quantum MAS NMR Spectroscopy in Dipolar Solids

A new pulse sequence for high-resolution solid-state heteronuclear double-quantum MAS NMR spectroscopy of dipolar-coupled spin-12 nuclei is introduced. It is based on the five-pulse sequence known from solution-state NMR, which is here applied synchronously to both spin species. The heteronuclear double-quantum (HeDQ) spinning-sideband patterns produced by this experiment are shown to be sensitive to the heteronuclear distance, as well as the relative orientations of the chemical-shift and dipolar tensors. In particular, it is shown that the HeDQ patterns exhibit an enhanced sensitivity to the chemical shielding tensors as compared with the single-quantum spinning-sideband patterns. The detection of HeDQ patterns via the I and S spins is discussed. The isolated (13)C-(1)H spin pair in deuterated ammonium formate with (13)C in natural abundance was chosen as a model system, and the perturbing influence of dipolar couplings to surrounding protons on the (13)C-(1)H DQ coherence is discussed. The pulse sequence can also be used as a heteronuclear double-quantum filter, hence providing information about heteronuclear couplings, and thus allowing the differentiation of quaternary and CH(n) bonded carbons. The elucidation of (13)C-(1)H dipolar proximities is presented for a sample of bisphenol A polycarbonate with (13)C in natural abundance, recorded with a broadband version of the synchronized five-pulse sequence.     

J-multiplied HSQC (MJ-HSQC): a new method for measuring 3J(HNHalpha) couplings in 15N-labeled proteins

A new method for the measurement of homonuclear 3J(HNHalpha) coupling constants in 15N-labeled small proteins is described. The method is based on a modified sensitivity enhanced HSQC experiment, where the 3J(HNHalpha) couplings are multiplied in the f1-dimension. The J-multiplication of homonuclear 3J(HNHalpha) couplings is based on simultaneous incrementation of 15N chemical shift and homonuclear coupling evolution periods. The time increment for the homonuclear coupling evolution period is chosen to be a suitable multiple (2N x t1) of the corresponding increment for 15N-shift evolution. This results in the splitting of the HSQC correlation in the f1-dimension by 2N x 3J(HNHalpha). Because the pulse sequence has good sensitivity and water suppression properties, it is particularly useful for natural abundance samples.     

Improved multiplicity-edited ADEQUATE experiments

A very simple strategy is proposed to extract carbon multiplicity information along with the classic knowledge of carbon-carbon connectivities in ADEQUATE experiments without affecting the sensitivity ratios of the original pulse schemes. These new multiplicity-edited ADEQUATE experiments prove to be highly helpful for complete 1H and 13C resonance assignment and also for automated and easy spin system characterization of samples at natural abundance, using a single NMR experiment.