Dynamics of Spin I=3/2 under Spin-Locking Conditions in an Ordered Environment

We have derived approximate analytic solutions to the master equation describing the evolution of the spin I=3/2 density operator in the presence of a radio-frequency (RF) field and both static and fluctuating quadrupolar interactions. Spectra resulting from Fourier transformation of the evolutions of the on-resonance spin-locked magnetization into the various coherences display two satellite pairs and, in some cases, a central line. The central line is generally trimodal, consisting of a narrow component related to a slowly relaxing mode and two broad components pertaining to two faster relaxing modes. The rates of the fast modes are sensitive to slow molecular motion. Neither the amplitude nor the width of the narrow component is affected by the magnitude of the static coupling, whereas the corresponding features of the broad components depend in a rather complicated manner on the spin-lock field strength and static quadrupolar interaction. Under certain experimental conditions, the dependencies of the amplitudes on the dynamics are seen to vanish and the relaxation rates reduce to relatively simple expressions. One of the promising emerging features is the fact that the evolutions into the selectively detected quadrupolar spin polarization order and the rank-two double-quantum coherence do not exhibit a slowly relaxing mode and are particularly sensitive to slow molecular motion. Furthermore, these coherences can only be excited in the presence of a static coupling and this makes it possible to discern nuclei in anisotropic from those in isotropic environment. The feasibility of the spin-lock pulse sequences with limited RF power and a nonvanishing average electric field gradient has been demonstrated through experiments on sodium in a dense lyotropic DNA liquid crystal.     

Tailored HCCH-TOCSY experiment for resonance assignment in the proximity of a paramagnetic center

The presence of a paramagnetic center may disturb both coherent and incoherent communication between nuclear spins that are affected, to some extent, by the hyperfine interaction. This is a limiting factor to an extensive use of paramagnetic probes in NMR spectroscopy to enhance partial alignment and to exploit cross correlation effects and pseudocontact shifts. We propose here an HCCH-TOCSY experiment tailored to identify spin systems involving resonances that are partly or completely affected by hyperfine interaction. The efficiency of polarization transfer steps when fast relaxing nuclei are involved is discussed. The sequence is tested for the protein Calbindin D(9k), in which one of the two native Ca2+ ions is replaced by the paramagnetic Ce3+ ion as well as for the oxidized form of cytochrome b(562).     

The effects of dissolved oxygen upon amide proton relaxation and chemical shift in a perdeuterated protein

The effects of dissolved molecular oxygen upon amide proton ((1)H(N)) longitudinal and transverse relaxation rates and chemical shifts were studied for a small protein domain, the second type 2 module of fibronectin ((2)F2)-isotopically enriched to 99% (2)H, 98% (15)N. Longitudinal relaxation rate enhancements, R(O(2))((1)H(N)), of individual backbone (1)H(N) nuclei varied up to 14 fold between a degassed and oxygenated (1 bar) solution, indicating that the oxygen distribution within the protein is inhomogeneous. On average, smaller relaxation rate enhancements were observed for (1)H(N) nuclei associated with the core of the protein compared to (1)H(N) nuclei closer to the surface, suggesting restricted oxygen accessibility to some regions. In agreement with an O(2)-(1)H(N) hyperfine interaction in the extreme narrowing limit, the (1)H(N) transverse relaxation rates showed no significant change, up to an oxygen pressure of 9.5 bar (the maximum pressure used in this study). For most (1)H(N) resonances, small deltadelta(O(2))((1)H(N)) hyperfine chemical shifts could be detected between oxygen pressures of 1 bar and 9.5 bar.     

Improved Broadband Inversion Performance for NMR in Liquids

New NMR broadband inversion pulses that compensate both for resonance offset and radiofrequency (RF) inhomogeneity are described. The approach described is a straightforward computer optimization of an initial digitized waveform generated from either a constant-amplitude frequency sweep or from an existing composite inversion pulse. Problems with convergence to local minima are alleviated by the way the optimization is carried out. For a given duration and maximum allowable RF field strength B₁ (but not necessarily given RMS power deposition), the resultant broadband inversion pulse (BIP) shows superior inversion compared to inversion pulses obtained from previous methods, including adiabatic inversion pulses. Any existing BIP can be systematically elaborated to build up longer inversion pulses that perform over larger and larger bandwidths. The resulting pulse need not be adiabatic throughout its duration or across the entire operational bandwidth.     

An Efficient NMR Experiment for Analyzing Sugar-Puckering in Unlabeled DNA: Application to the 26-kDa Dead Ringer–DNA Complex

We present a new NMR experiment for estimating the type and degree of sugar-puckering in high-molecular-weight unlabeled DNA molecules. The experiment consists of a NOESY sequence preceded by a constant-time scalar coupling period. Two subexperiments are compared, each differing in the amount of time the (3)J(H3'H2') and (3)J(H3'H2") couplings are active on the H3' magnetization. The resultant data are easy to analyze, since a comparison of the signal intensities of any resolved NOE cross peak originating from H3' atoms of the duplex can be used to estimate the sum of the (3)J(H3'H2') and (3)J(H3'H2") couplings and thus the puckering type of the deoxyribose ring. Isotope filters to eliminate signals of the (13)C-labeled component in the F1-dimension are implemented, facilitating analyses of high-molecular-weight protein-DNA complexes containing (13)C-labeled protein and unlabeled DNA. The utility of the experiment is demonstrated on the 26-kDa Dead Ringer protein-DNA complex and reveals that the DNA uniformly adopts the S-type configuration when bound to protein.     

Spin-lattice relaxation and a fast T1-map acquisition method in MRI with transient-state magnetization

The magnetization under the spin-lattice relaxation and the nuclear magnetic resonance radiofrequency (RF) pulses is calculated for a signal RF pulse train and for a sequence of multiple RF pulse-trains. It is assumed that the transverse magnetization is zero when each RF pulse is applied. The result expressions can be grouped into two terms: a decay term, which is proportional to the initial magnetization M0, and a recovery term, which has no M0 dependence but strongly depends on the spin-lattice relaxation and the equilibrium magnetization Meq. In magnetic resonance pulse sequences using magnetization in transient state, the recovery term produces artifacts and can seriously degrade the function of the preparation sequence for slice selection, contrast weighting, phase encoding, etc. This work shows that the detrimental effect can be removed by signal averaging in an eliminative fashion. A novel fast data acquisition method for constructing the spin-lattice relaxation (T1) map is introduced. The method has two features: (i) By using eliminative averaging, the curve to fit the T1 value is a decay exponential function rather than a recovery one as in conventional techniques; therefore, the measurement of Meq is not required and the result is less susceptible to the accuracy of the inversion RF pulse. (ii) The decay exponential curve is sampled by using a sequence of multiple pulse-trains. An image is reconstructed from each train and represents a sample point of the curve. Hence a single imaging sequence can yield multiple sample points needed for fitting the T1 value in contrast to conventional techniques that require repeating the imaging sequence for various delay values but obtain only one sample point from each repetition.     

T1–T2 Correlation Spectra Obtained Using a Fast Two-Dimensional Laplace Inversion

Spin relaxation is a sensitive probe of molecular structure and dynamics. Correlation of relaxation time constants, such as T₁ and T₂, conceptually similar to the conventional multidimensional spectroscopy, have been difficult to determine primarily due to the absense of an efficient multidimensional Laplace inversion program. We demonstrate the use of a novel computer algorithm for fast two-dimensional inverse Laplace transformation to obtain T₁–T₂ correlation functions. The algorithm efficiently performs a least-squares fit on two-dimensional data with a nonnegativity constraint. We use a regularization method to find a balance between the residual fitting errors and the known noise amplitude, thus producing a result that is found to be stable in the presence of noise. This algorithm can be extended to include functional forms other than exponential kernels. We demonstrate the performance of the algorithm at different signal-to-noise ratios and with different T₁–T₂ spectral characteristics using several brine-saturated rock samples.     

Time-optimal coherence-order-selective transfer of in-phase coherence in heteronuclear IS spin systems

Based on principles of geometric optimal control theory, coherence transfer building blocks can be derived which achieve optimal sensitivity. Here, experimental pulse sequences are presented that achieve the best possible coherence-order-selective in-phase transfer (S(-)-->I(-)) for a heteronuclear 2-spin system for any given mixing time in the absence of relaxation. For short mixing times, the optimal experiment improves the sensitivity of isotropic mixing by up to 12.5%.     

Imaging the long-range dipolar field in structured liquid state samples

We describe imaging experiments in which the pattern of the dipolar field generated by spatially modulated nuclear magnetization is directly visualized in simply structured phantoms. Two types of experiment have been carried out at 11.7 T using (1)H NMR signals. In the first, the field from a single spin species is imaged via its own NMR signal. In the second, the NMR signal from one spin species is used to image the field generated by a second species. The field patterns measured in these experiments correspond well with those calculated using simple theoretical expressions for the dipolar field. The results also directly demonstrate the spatial sensitivity of the signal generated using dipolar field effects, indicating that the range of the field depends upon the inverse of the spatial frequency with which the magnetization is modulated.     

Dipolar and J encoded DQ MAS spectra under rotational resonance

A two-dimensional (2D) double-quantum (DQ) experiment under rotational resonance (R(2)) conditions is introduced for evaluating dipolar couplings in rotating solids. The contributions from the R(2)-recoupled dipolar interaction and the J coupling can be conveniently separated in the resulting 2D R(2)-DQ spectrum, so that the unknown dipolar coupling can readily be extracted, provided that the values of the involved J coupling constants are known. Since the measured parameters are integral intensity ratios between suitably chosen absorption peaks in the 2D spectrum, the proposed method is characterized by a reduced sensitivity to relaxation parameters. The effect of rotor-modulated terms, including chemical shift anisotropy, is efficiently averaged out by synchronizing the excitation/reconversion time with the rotor period. All of these features are demonstrated theoretically by the example of two model systems, namely, isolated spin-pairs and a three-spin system. The results of the theoretical models are applied to both (13)C and (1)H nuclei to extract dipolar couplings in uniformly (13)C labeled L-alanine and a crosslinked natural rubber.     

H Spectroscopy without Solvent Suppression: Characterization of Signal Modulations at Short Echo Times

While most proton (¹H) spectra acquired in vivo utilize selective suppression of the solvent signal for more sensitive detection of signals from the dilute metabolites, recent reports have demonstrated the feasibility and advantages of collecting in vivo data without solvent attenuation. When these acquisitions are performed at short echo times, the presence of frequency modulations of the water resonance may become an obstacle to the identification and quantitation of metabolite resonances. The present report addresses the characteristics, origin, and elimination of these sidebands. Sideband amplitudes were measured as a function of delay time between gradient pulse and data collection, as a function of gradient pulse amplitude, and as a function of spatial location of the sample for each of the three orthogonal gradient sets. Acoustic acquisitions were performed to demonstrate the correlation between mechanical vibration resonances and the frequencies of MR sidebands. A mathematical framework is developed and compared with the experimental results. This derivation is based on the theory that these frequency modulations are induced by magnetic field fluctuations generated by the transient oscillations of gradient coils.     

Direct measurement of dipole-dipole/CSA cross-correlated relaxation by a constant-time experiment

Relaxation rates in NMR are usually measured by intensity modulation as a function of a relaxation delay during which the relaxation mechanism of interest is effective. Other mechanisms are often suppressed during the relaxation delay by pulse sequences which eliminate their effects, or cancel their effects when two data sets with appropriate combinations of relaxation rate effects are added. Cross-correlated relaxation (CCR) involving dipole-dipole and CSA interactions differ from auto-correlated relaxation (ACR) in that the signs of contributions can be changed by inverting the state of one spin involved in the dipole-dipole interaction. This property has been exploited previously using CPMG sequences to refocus CCR while ACR evolves. Here we report a new pulse scheme that instead eliminates intensity modulation by ACR and thus allows direct measurement of CCR. The sequence uses a constant time relaxation period for which the contribution of ACR does not change. An inversion pulse is applied at various points in the sequence to effect a decay that depends on CCR only. A 2-D experiment is also described in which chemical shift evolution in the indirect dimension can share the same constant period. This improves sensitivity by avoiding the addition of a separate indirect dimension acquisition time. We illustrate the measurement of residue specific CCR rates on the non-myristoylated yeast ARF1 protein and compare the results to those obtained following the conventional method of measuring the decay rates of the slow and fast-relaxing (15)N doublets. The performances of the two methods are also quantitatively evaluated by simulation. The analysis shows that the shared constant-time CCR (SCT-CCR) method significantly improves sensitivity.     

Simultaneous selective detection of multiple quantum spectra

A three-dimensional multiple-quantum NMR experiment that produces individual spectra of all quantum orders is described. The separation of different quantum orders is accomplished via Fourier transformation with respect to the phase of the first two pulses of a generic three-pulse multiple-quantum sequence. This dramatically reduces the time required to obtain several selectively detected spectra and enhances the sensitivity and digital resolution from that obtained using the original two-dimensional technique. The experiment is demonstrated on the protons of para-chlorotoluene dissolved in the nematic liquid crystal Merck ZLI-1132.     

Double-quantum dipolar recoupling at high magic-angle spinning rates

A full investigation of the possible homonuclear double-quantum recoupling sequences, based on the RN family of sequences with N < or = 20, is given. Several new RN sequences, R16(6)(5), R18(8)(5), and R18(10)(5), were applied at high magic-angle spinning rates and compared with theory. The R18(10)(5) technique can be used to recouple dipolar couplings at spinning rates up to 39 kHz, and the application of the sequence in an INADEQUATE experiment is shown for a spinning rate of 30 kHz.     

Changes in Porcine Muscle Water Characteristics during Growth—An in Vitro Low-Field NMR Relaxation Study

This study investigates the effects of developmental stage and muscle type on the mobility and distribution of water within skeletal muscles, using low-field ¹H-NMR transverse relaxation measurements in vitro on four different porcine muscles (M. longissimus dorsi, M. semitendinosus, M. biceps femoris, M. vastus intermedius) from a total of 48 pigs slaughtered at various weight classes between 25 kg and 150 kg. Principal component analysis (PCA) revealed effects of both slaughter weight and muscle type on the transverse relaxation decay. Independent of developmental stage and muscle type, distributed exponential analysis of the NMR T₂ relaxation data imparted the existence of three distinct water populations, T_2b, T₂₁, and T₂₂, with relaxation times of approximately 1–10, 45–120, and 200–500 ms, respectively. The most profound change during muscle growth was a shift toward faster relaxation in the intermediate time constant, T₂₁. It decreased by approx. 24% in all four muscle types during the period from 25 to 150 kg live weight. Determination of dry matter, fat, and protein content in the muscles showed that the changes in relaxation time of the intermediate time constant, T₂₁, during growth should be ascribed mainly to a change in protein content, as the protein content explained 77% of the variation in the T₂₁ time constant. Partial least squares (PLS) regression revealed validated correlations in the region of 0.58 to 0.77 between NMR transverse relaxation data and muscle development for all the four muscle types, which indicates that NMR relaxation measurements may be used in the prediction of muscle developmental stage.     

MUSIC and Aromatic Residues: Amino Acid Type-Selective H–N Correlations, III

Amino acid type-selective experiments can help to remove ambiguities in automated assignment procedures for ¹⁵N/¹³C-labeled proteins. Here we present five triple-resonance experiments that yield amino acid type-selective ¹H–¹⁵N correlations for aromatic amino acids. Four of the novel experiments are based on the MUSIC coherence transfer scheme that replaces the initial INEPT transfer and is selective for CH₂. The MUSIC sequence is combined with selective excitation pulses to create experiments for Trp (W-HSQC) as well as Phe, Tyr, and His (FYH-HSQC). In addition, an experiment selective for Trp H¹–N¹ is presented. The new experiments are recorded as two-dimensional experiments and their performance is demonstrated with the application to a protein domain of 115 amino acids.     

Application of high-resolution magic-angle spinning NMR spectroscopy to define the cell uptake of MRI contrast agents

A new method, based on proton high-resolution magic-angle spinning ((1)H HR-MAS) NMR spectroscopy, has been employed to study the cell uptake of magnetic resonance imaging contrast agents (MRI-CAs). The method was tested on human red blood cells (HRBC) and white blood cells (HWBC) by using three gadolinium complexes, widely used in diagnostics, Gd-BOPTA, Gd-DTPA, and Gd-DOTA, and the analogous complexes obtained by replacing Gd(III) with Dy(III), Nd(III), and Tb(III) (i.e., complexes isostructural to the ones of gadolinium but acting as shift agents). The method is based on the evaluation of the magnetic effects, line broadening, or induced lanthanide shift (LIS) caused by these complexes on NMR signals of intra- and extracellular water. Since magnetic effects are directly linked to permeability, this method is direct. In all the tests, these magnetic effects were detected for the extracellular water signal only, providing a direct proof that these complexes are not able to cross the cell membrane. Line broadening effects (i.e., the use of gadolinium complexes) only allow qualitative evaluations. On the contrary, LIS effects can be measured with high precision and they can be related to the concentration of the paramagnetic species in the cellular compartments. This is possible because the HR-MAS technique provides the complete elimination of bulk magnetic susceptibility (BMS) shift and the differentiation of extra- and intracellular water signals. Thus with this method, the rapid quantification of the MRI-CA amount inside and outside the cells is actually feasible.     

Improved Spin-Echo-Edited NMR Diffusion Measurements

The need for simple and robust schemes for the analysis of ligand-protein binding has resulted in the development of diffusion-based NMR techniques that can be used to assay binding in protein solutions containing a mixture of several ligands. As a means of gaining spectral selectivity in NMR diffusion measurements, a simple experiment, the gradient modified spin-echo (GOSE), has been developed to reject the resonances of coupled spins and detect only the singlets in the (1)H NMR spectrum. This is accomplished by first using a spin echo to null the resonances of the coupled spins. Following the spin echo, the singlet magnetization is flipped out of the transverse plane and a dephasing gradient is applied to reduce the spectral artifacts resulting from incomplete cancellation of the J-coupled resonances. The resulting modular sequence is combined here with the BPPSTE pulse sequence; however, it could be easily incorporated into any pulse sequence where additional spectral selectivity is desired. Results obtained with the GOSE-BPPSTE pulse sequence are compared with those obtained with the BPPSTE and CPMG-BPPSTE experiments for a mixture containing the ligands resorcinol and tryptophan in a solution of human serum albumin.