Rapid 3D NMR using the filter diagonalization method: application to oligosaccharides derivatized with 13C-labeled acetyl groups

Rapid 3D NMR spectroscopy of oligosaccharides having isotopically labeled acetyl "isotags" was made possible with high resolution in the indirect dimensions using the filter diagonalization method (FDM). A pulse sequence was designed for the optimal correlation of acetyl methyl protons, methyl carbons, and carbonyl carbons. The multi-dimensional nature of the FDM, coupled with the advantages of constant-time evolution periods, resulted in marked improvements over Fourier transform (FT) and mirror-image linear prediction (MI-LP) processing methods. The three methods were directly compared using identical data sets. A highly resolved 3D spectrum was achieved with the FDM using a very short experimental time (28 min).     

High-resolution four-dimensional carbon-correlated 1H-1H ROESY experiments employing isotags and the filter diagonalization method for effective assignment of glycosidic linkages in oligosaccharides

Four-dimensional nuclear magnetic resonance spectroscopy of oligosaccharides that correlates 1H-1H ROESY cross peaks to two additional 13C frequency dimensions is reported. The 13C frequencies were introduced by derivatization of all free hydroxyl groups with doubly 13C-labeled acetyl isotags. Pulse sequences were optimized for processing with the filter diagonalization method. The extensive overlap typically observed in 2D ROESY 1H-1H planes was alleviated by resolution of ROESY cross peaks in the two added dimensions associated with the carbon frequencies of the isotags. This enabled the interresidue 1H-1H ROESY cross peaks to be unambiguously assigned hence spatially proximate sugar spin systems across glycosidic bonds could be effectively ascertained. An experiment that selectively amplifies interresidue ROESY 1H-1H cross peaks is also reported. It moves the magnetization of an intraresidue proton normally correlated to a sugar H-1 signal orthogonally along the z axis prior to a Tr-ROESY mixing sequence. This virtually eliminates the incoherent intraresidue ROESY transfer, suppresses coherent TOCSY transfer, and markedly enhances the intensity of interresidue ROESY cross peaks.     

Enhanced spectral resolution by high-dimensional NMR using the filter diagonalization method and "hidden" dimensions

High-dimensional (HD) NMR spectra have poorer digital resolution than low-dimensional (LD) spectra, for a fixed amount of experiment time. This has led to "reduced-dimensionality" strategies, in which several LD projections of the HD NMR spectrum are acquired, each with higher digital resolution; an approximate HD spectrum is then inferred by some means. We propose a strategy that moves in the opposite direction, by adding more time dimensions to increase the information content of the data set, even if only a very sparse time grid is used in each dimension. The full HD time-domain data can be analyzed by the filter diagonalization method (FDM), yielding very narrow resonances along all of the frequency axes, even those with sparse sampling. Integrating over the added dimensions of HD FDM NMR spectra reconstitutes LD spectra with enhanced resolution, often more quickly than direct acquisition of the LD spectrum with a larger number of grid points in each of the fewer dimensions. If the extra-dimensions do not appear in the final spectrum, and are used solely to boost information content, we propose the moniker hidden-dimension NMR. This work shows that HD peaks have unmistakable frequency signatures that can be detected as single HD objects by an appropriate algorithm, even though their patterns would be tricky for a human operator to visualize or recognize, and even if digital resolution in an HD FT spectrum is very coarse compared with natural line widths.     

Progress on the two-dimensional filter diagonalization method. An efficient doubling scheme for two-dimensional constant-time NMR

An efficient way to treat two-dimensional (2D) constant-time (CT) NMR data using the filter diagonalization method (FDM) is presented. In this scheme a pair of N- and P-type data sets from a 2D CT NMR experiment are processed jointly by FDM as a single data set, twice as large, in which the signal effectively evolves in time for twice as long. This scheme is related to "mirror-image" linear prediction, but with the distinction that the data are directly used, without any preprocessing such as Fourier transformation along one dimension, or point-by-point reflection. As the signal has nearly perfect Lorentzian line shape in the CT dimension, it can be efficiently handled by the FDM approach. Applied to model and experimental signals, the scheme shows significant resolution improvement, and appears to tolerate noise reasonably well. Other complex aspects of multidimensional FDM are discussed and illustrated.     

Suppression of sampling artefacts in high-resolution four-dimensional NMR spectra using signal separation algorithm

The development of non-uniform sampling (NUS) strategies permits to obtain high-dimensional spectra with increased resolution in significantly reduced experimental time. We extended a previously proposed signal separation algorithm (SSA) to process sparse four-dimensional NMR data. It is employed for two experiments carried out for a partially unstructured 114-residue construct of chicken Engrailed 2 protein, namely 4D HCCH-TOCSY and 4D C,N-edited NOESY. The SSA allowed us to obtain high-quality spectra using only as little as 0.16% of the available samples, with low sampling artefacts approaching the thermal noise level in most spectral regions. It is demonstrated that NUS 4D HCCH-TOCSY is dominated by sampling noise and requires efficient artefact suppression. On the other hand, 4D C,N-edited NOESY is a particularly attractive experiment for NUS, as the absence of diagonal peaks renders the problem of artefacts less critical. We also present a transverse-relaxation optimized sequence for HMQC that is especially designed for longer evolution periods in the indirectly detected proton dimension in high-dimensional pulse sequences. In conjunction with novel sampling strategies and efficient processing methods, this improvement enabled us to obtain unique structural information about aliphatic-amide contacts.     

Regularization of the two-dimensional filter diagonalization method: FDM2K

We outline an important advance in the problem of obtaining a two-dimensional (2D) line list of the most prominent features in a 2D high-resolution NMR spectrum in the presence of noise, when using the Filter Diagonalization Method (FDM) to sidestep limitations of conventional FFT processing. Although respectable absorption-mode spectra have been obtained previously by the artifice of “averaging” several FDM calculations, no 2D line list could be directly obtained from the averaged spectrum, and each calculation produced numerical artifacts that were demonstrably inconsistent with the measured data, but which could not be removed a posteriori. By regularizing the intrinsically ill-defined generalized eigenvalue problem that FDM poses, in a particular quite plausible way, features that are weak or stem from numerical problems are attenuated, allowing better characterization of the dominant spectral features. We call the new algorithm FDM2K.     

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.     

Solvent-localized NMR spectroscopy using the distant dipolar field: a method for NMR separations with a single gradient

Solvent-localized NMR (SOLO) is a new method which allows the separation of NMR spectra of substances dissolved in different solvents. It uses the selective HOMOGENIZED pulse sequence to produce a two-dimensional NMR spectrum resulting from intermolecular zero-quantum coherences in one distinct solvent. The detected signal is locally refocused by the action of the distant dipolar field, which is created by a frequency selective pulse only in regions containing the selected solvent. The prerequisites are that the different solvents have sufficiently different chemical shifts to be excited separately and that compartments with different solvents are spatially separated by more than the typical diffusion distance. Here, the method is demonstrated for the solvents water and DMSO on a length scale of 0.5 mm. Because signal in the spectra is refocused locally, SOLO is insensitive to variations in the magnetic field which may result from inhomogeneities or structures in the sample. This makes applications in strongly structured samples possible. SOLO is the first method that achieves localization of NMR signal with a single gradient pulse. Therefore, it can be used in conventional NMR spectrometers with one-axis gradient systems and lends itself to a wide range of applications including in vivo NMR.     

A new method for rapid realization of the high-resolution extended-range holographic image-deblurring filter

The filter is photographically realized for its amplitude by two sandwiched components of combined extended-dynamic-range slope γ=1, and for its phase by a high-spatial-frequency range hologram recorded through them in a “triple-diffraction” arrangement.     

Toroid cavity detectors for high-resolution NMR spectroscopy and rotating frame imaging: capabilities and limitations

The capabilities of toroid cavity detectors for simultaneous rotating frame imaging and NMR spectroscopy have been investigated by means of experiments and computer simulations. The following problems are described: (a) magnetic field inhomogeneity and subsequent loss of chemical shift resolution resulting from bulk magnetic susceptibility effects, (b) image distortions resulting from off-resonance excitation and saturation effects, and (c) distortion of lineshapes and images resulting from radiation damping. Also, special features of signal analysis including truncation effects and the propagation of noise are discussed. B(0) inhomogeneity resulting from susceptibility mismatch is a serious problem for applications requiring high spectral resolution. Image distortions resulting from off-resonance excitation are not serious within the rather narrow spectral range permitted by the RF pulse lengths required to read out the image. Incomplete relaxation effects are easily recognized and can be avoided. Also, radiation damping produces unexpectedly small effects because of self-cancellation of magnetization and short free induction decay times. The results are encouraging, but with present designs only modest spectral resolution can be achieved.     

TRAPDOR double-resonance and high-resolution MAS NMR for structural and template studies in zeolite ZSM-5

A combination of ²⁷Al magic-angle spinning (MAS)/multiple-quantum (MQ) MAS, and ²⁷Al–{¹⁴N} TRAnsfer of Population in DOuble-Resonance (TRAPDOR) nuclear magnetic resonance (NMR) was used to study aluminium environments in zeolite ZSM-5. ²⁷Al–{¹⁴N} TRAPDOR experiments, in combination with ¹⁴N NMR were employed to show that the two tetrahedral peaks observed in the ²⁷Al MAS/3Q-MAS spectra of as-synthesized ZSM-5 are due to aluminium atoms occupying crystallographically inequivalent T-sites. A ¹³C–{²⁷Al} TRAPDOR experiment was used to study the template, tetrapropyl ammonium bromide (TPABr), in the three-dimensional pore system of ZSM-5. The inequivalency of the methyl groups of TPA was observed in the ¹³C–{²⁷Al} TRAPDOR spectra of as-synthesized ZSM-5 and the motion of the methyl end of the propyl chain appeared to be more restricted in the sinusoidal channel than in the straight channel.     

Indirect covariance NMR spectroscopy of through-bond homo-nuclear correlations for quadrupolar nuclei in solids under high-resolution

Indirect covariance NMR spectroscopy is demonstrated in solids, and we show that it can be used to obtain through-bond 2D homo-nuclear correlation spectra for quadrupolar nuclei under high-resolution. These spectra, generated with indirect covariance from a hetero-nuclear correlation spectrum, are equivalent to those recorded with the through-bond homo-nuclear hetero-nuclear single-quantum correlation (H-HSQC) method very recently proposed. However, the indirect covariance method can save a lot of experiment time, compared to the H-HSQC experiments, which allows introducing a high-resolution quadrupolar filter, thus providing a much better resolution, even on medium-field spectrometers. The covariance concept can be used to generate many different "indirectly-detected" high-resolution homo-nuclear correlation spectra with through-space or through-bond correlations for spin 1/2 or quadrupolar nuclei. We also propose a simple method that decreases the noise in all (direct or indirect) covariance methods.     

Use of SPAM and FAM pulses in high-resolution MAS NMR spectroscopy of quadrupolar nuclei

The merits of SPAM and FAM pulses for enhancing the conversion of triple- to single-quantum coherences in the two-dimensional MQMAS experiment are compared using (87)Rb (spin I=3/2) and (27)Al (I=5/2) NMR of crystalline and amorphous materials. Although SPAM pulses are more easily optimized, our experiments and simulations suggest that FAM pulses yield greater signal intensity in all cases. In conclusion, we argue that, as originally suggested, SPAM and FAM pulses are best implemented in phase-modulated whole-echo MQMAS experiments and that the use of SPAM pulses to record separate echo and antiecho data sets, which are then combined, generally yields lower signal-to-noise ratios.     

Another approach to protons with constricted mobility in white matter: pilot studies using wideline and high-resolution NMR spectroscopy

We report a new approach for the identification of an independent method of studying the semi-solid pool of protons, i.e., protons with constrained motion as a result of being bound to lipid and protein matrices. These protons cannot be observed using conventional imaging techniques since their transverse relaxation times are much shorter than the minimum echo times that are currently available on clinical scanners. In this pilot study, in vitro multicomponent transverse relaxation experiments were made on human white matter slices, fixed in formalin (7 normal and 5 with multiple sclerosis). The transverse relaxation decay curves were multiexponential and were decomposed to yield three primary components. The shortest T(2) component that we obtained (a component too short to be seen by in vivo methods) was of the order of microseconds. We hypothesize that this might correspond to the macromolecular pool of lipid protons trapped within the myelin sheaths. To our knowledge, this is the first attempt at extracting this ultra short T(2) component from human white matter. Subsequently, an attempt was made to directly detect the lipid protons in a proton NMR spectrum and, if possible, measure their concentration in some of the tissues, using the technique of magic angle spinning.     

Perspectives of solution NMR spectroscopy for structural and functional studies of integral membrane proteins

This article discusses future perspectives of solution NMR spectroscopy to study structures and functions of integral membrane proteins at atomic resolution, based on a review of recent progress in this area. Several selected examples of structure determinations, as well as functional studies of integral membrane proteins are highlighted. We expect NMR spectroscopy to make future key scientific contributions to understanding membrane protein function, in particular for large membrane protein systems with known three-dimensional structure. Such situations can benefit from the fact that functional NMR studies have substantially less limitations by molecular size than a full de novo structure determination. Therefore, the general potential for NMR spectroscopy to solve biologic key questions associated with integral membrane proteins is very promising.