Spatially Selective Heteronuclear Multiple‐Quantum Coherence Spectroscopy for Biomolecular NMR Studies

Spatially selective heteronuclear multiple‐quantum coherence (SS HMQC) NMR spectroscopy is developed for solution studies of proteins. Due to “time‐staggered” acquisitioning of free induction decays (FIDs) in different slices, SS HMQC allows one to use long delays for longitudinal nuclear spin relaxation at high repetition rates. To also achieve high intrinsic sensitivity, SS HMQC is implemented by combining a single spatially selective ¹H excitation pulse with nonselective ¹H 180° pulses. High‐quality spectra were obtained within 66 s for a 7.6 kDa uniformly ¹³C,¹⁵N‐labeled protein, and within 45 and 90 s for, respectively, two proteins with molecular weights of 7.5 and 43 kDa, which were uniformly ²H,¹³C,¹⁵N‐labeled, except for having protonated methyl groups of isoleucine, leucine and valine residues.     

NMR quantification of trace components in complex matrices by band‐selective excitation with adiabatic pulses

The use of band‐selective excitation with adiabatic pulses to rapidly obtain NMR spectra of trace components in the presence of strong signals is described, along with qualitative and quantitative examples from food matrices like olive oil and honey. Copyright © 2009 John Wiley & Sons, Ltd.     

Frequency‐Switched Single‐Transition Cross‐Polarization: A Tool for Selective Experiments in Biomolecular NMR

Frequency‐switched single‐transition cross‐polarization (FS‐ST‐CP) provides a versatile tool for selective coherence transfer in heteronuclear NMR of biomolecules such as proteins and nucleic acids. This type of coherence transfer is spin‐state‐selective and can therefore benefit from the extension of the life‐times of selected coherences due to partial cancellation of interfering relaxation mechanisms. The limits of the selectivity of the transfer are discussed by theory and illustrated by experiment. The methods are particularly efficient to obtain quantitative structural and dynamic information for selected residues in medium‐sized nitrogen‐15 or carbon‐13 labeled macromolecules.     

Pivotal steps towards quantification of molecular diffusion coefficients by NMR.

Using nuclear magnetic resonance (NMR) spectroscopy with a pair of pulsed field gradients (PFGs), Stajeskal and Tanner successfully measured molecular diffusion coefficients in solution in 1965. This method has since been used extensively in various applications, especially after the PFG was implemented in commercial NMR probes. Due to the nonuniformity of the PFG and radio frequency (RF) fields, molecules distributed throughout the sample experience different PFG and RF fields and contribute unevenly to the measured diffusion coefficients, resulting in considerable errors in conventional NMR diffusion experiments. By selective excitation of a central sample region with an offset-independent adiabatic inversion pulse and a PFG, a uniform RF field can be assumed, and the PFG can be represented as a linear approximation. Under these conditions, the molecules diffuse as if they were all experiencing the same effective gradient g(e), leading to a Gaussian signal decay as a function of the PFG strength. Quantitative measurement of molecular diffusion coefficients is therefore made possible. From the diffusion coefficient of a 90 % H(2)O/10 % D(2)O sample, it is convenient to calibrate g(e) with a Java program. In a similar way the nonlinearity of the PFG can be corrected.     

Adiabatic JHSQC for 13C isotopomer analysis

A previous implementation of JHSQC enhanced the detection limits for 13C isotopomer analysis, but owing to experimental instabilities required extensive calibration prior to data collection. The addition of adiabatic pulses as well as a double spin-echo during the heteronuclear transfer periods has dramatically increased the precision of the experiment and done away with the need for calibration prior to running each sample. This new experiment allows the sensitivity gains from inverse detection to be realized in a time-efficient manner for the experimentalist.     

Longitudinal Relaxation Enhancement in 1H NMR Spectroscopy of Tissue Metabolites via Spectrally Selective Excitation

Nuclear magnetic resonance spectroscopy is governed by longitudinal (T₁) relaxation. For protein and nucleic acid experiments in solutions, it is well established that apparent T₁ values can be enhanced by selective excitation of targeted resonances. The present study explores such longitudinal relaxation enhancement (LRE) effects for molecules residing in biological tissues. The longitudinal relaxation recovery of tissue resonances positioned both down‐ and upfield of the water peak were measured by spectrally selective excitation/refocusing pulses, and compared with conventional water‐suppressed, broadband‐excited counterparts at 9.4 T. Marked LRE effects with up to threefold reductions in apparent T₁ values were observed as expected for resonances in the 6–9 ppm region; remarkably, statistically significant LRE effects were also found for several non‐exchanging metabolite resonances in the 1–4 ppm region, encompassing 30–50 % decreases in apparent T₁ values. These LRE effects suggest a novel means of increasing the sensitivity of tissue‐oriented experiments, and open new vistas to investigate the nature of interactions among metabolites, water and macromolecules at a molecular level.     

Selective 1D HCH experiment: a fast NMR tool that connect protons belonging to different spin systems

A selective 1D version of the HCH experiment (selHCH) is proposed for the efficient and fast correlation between protons belonging to different spin systems. The experiment consists of two consecutive, doubly selective heteronuclear J(CH) transfer steps that can individually be optimized. As any conventional proton-selective 1D experiment, the successful application of a frequency-selective 180° pulse on a well-isolated proton is the only practical requirement. The resulting clean selHCH spectrum probes to be an excellent complement to the conventional selTOCSY experiment to trace out proton-proton J connectivities through transparent non-protonated carbons or heteroatom centers. Several selHCH examples on small molecules are provided showing the improvements with respect to the selTOCSY experiment.     

Fully Resolved NMR Correlation Spectroscopy

A new correlation experiment cited as “push‐G‐SERF” is reported. In the resulting phased 2D spectrum, the chemical shift information is selected along the direct dimension, whereas scalar couplings involving a selected proton nucleus are edited in the indirect domain. The robustness of this pulse sequence is demonstrated on compounds with increasing structural and spectral complexity, using state‐of‐the‐art spectrometers. It allows for full resolution of both dimensions of the spectrum, yielding a straightforward assignment and measurement of the coupling network around a given proton in the molecule. This experiment is intended for chemists who want to address efficiently the structural analysis of molecules with an overcrowded spectrum.     

Accelerated NMR Spectroscopy with Low‐Rank Reconstruction

Accelerated multi‐dimensional NMR spectroscopy is a prerequisite for high‐throughput applications, studying short‐lived molecular systems and monitoring chemical reactions in real time. Non‐uniform sampling is a common approach to reduce the measurement time. Here, a new method for high‐quality spectra reconstruction from non‐uniformly sampled data is introduced, which is based on recent developments in the field of signal processing theory and uses the so far unexploited general property of the NMR signal, its low rank. Using experimental and simulated data, we demonstrate that the low‐rank reconstruction is a viable alternative to the current state‐of‐the‐art technique compressed sensing. In particular, the low‐rank approach is good in preserving of low‐intensity broad peaks, and thus increases the effective sensitivity in the reconstructed spectra.     

Strategies for 1H-Detected Dynamic Nuclear Polarization Magic-Angle Spinning NMR Spectroscopy

Combining dynamic nuclear polarization with proton detection significantly enhances the sensitivity of magic-angle spinning NMR spectroscopy. Herein, the feasibility of proton-detected experiments with slow (10 kHz) magic angle spinning was demonstrated. The improvement in sensitivity permits the acquisition of indirectly detected ¹⁴N NMR spectra allowing biomolecular structures to be characterized without recourse to isotope labelling. This provides a new tool for the structural characterization of environmental and medical samples, in which isotope labelling is frequently intractable.     

Broadband and band-selective IMPRESS-gHMBC: compensation of refocusing inefficiency with synchronized inversion sweep

Compensation of refocusing inefficiency in a gHMBC experiment by replacing the rectangular pi pulse with a pair of adiabatic pulses with synchronized inversion sweep (CRISIS) significantly improves the performance of the gHMBC experiment. The CRISIS-gHMBC experiment retains the pure absorptive shapes in F1 and hence results in better lineshape and higher resolution than the current versions of magnitude mode gHMBC spectra. When used as a broadband experiment, CRISIS-gHMBC, owing to better refocusing efficiency of the adiabatic pulse pairs, gives improved performance across the 13C spectral width. Moreover, it is shown that CRISIS-gHMBC is a robust and improved alternative and when used along with the IMPRESS (Improved Resolution using Symmetrically Shifted pulses) technique further increases the sensitivity and resolution without additional experimental time. The IMPRESS-CRISIS combination is demonstrated for broadband gHMBC and band-selective gHMBC experiments. The ICbs-gHMBC [IMPRESS-CRISIS-band-selective gHMBC] experiment is an attractive and better alternative to individual band-selective gHMBC.     

New DEFT sequences for the acquisition of one-dimensional carbon NMR spectra of small unlabelled molecules

The acquisition time and quality of 1D 13C{1H} spectra can be improved substantially by using a modified driven equilibrium Fourier transform (DEFT) sequence, which is specifically designed to compensate for the effects of B1 inhomogeneity, pulse miscalibration and frequency offsets. The new sequence, called uniform driven equilibrium Fourier transform (UDEFT), returns the carbon magnetization with a high accuracy along its equilibrium position after each transient is complete. Thus, the sequence allows the use of relaxation delays (RD), which are much shorter than the carbon T1 of the molecule, thereby speeding up the acquisition process of 1D 13C{1H} spectra. To achieve this level of performance, UDEFT employs a refocusing element constituted by a composite adiabatic carbon pulse surrounded by two 90 degrees carbon pulses whose phases are designed to compensate for 90 degrees pulse miscalibrations in an MLEV manner (90 degrees+x-tau(FID)-180+y(Adia)-tau-90 degrees+x-180 degrees+x(Adia)). A version of the UDEFT sequence allows recording 1D 13C{1H} spectra devoid of heteronuclear NOE by using a matched adiabatic 1H decoupling scheme where an even number of 180 degrees adiabatic pulses is applied during the UDEFT module. Spectra of a solution of 300 mM camphor that contains some carbon nuclei with very long T1 relaxation times (90 s and 78 s) were acquired with 128 scans in 10 min using a 5 s relaxation delay.     

利用选择性激发核磁共振对混合物中的两种化合物进行结构解析

利用现代NMR的1D、2D技术对一个混合物进行了分析。结果表明：混合物由两种化合物组成。为了同时准确确定两种化合物的结构，本工作应用了1D-TOCSY技术，利用该技术选择性强的特点来补充常规的1D、2DHMR实验所提供的分子结构的信息。在没有进行预分离的条件下，顺利地完成了样品中两种化合物的核磁信号归属，并最终确定了它们的结构。     

Labeling Strategy and Signal Broadening Mechanism of Protein NMR Spectroscopy in Xenopus laevis Oocytes

We used Xenopus laevis oocytes, a paradigm for a variety of biological studies, as a eukaryotic model system for in‐cell protein NMR spectroscopy. The small globular protein GB1 was one of the first studied in Xenopus oocytes, but there have been few reports since then of high‐resolution spectra in oocytes. The scarcity of data is at least partly due to the lack of good labeling strategies and the paucity of information on resonance broadening mechanisms. Here, we systematically evaluate isotope enrichment and labeling methods in oocytes injected with five different proteins with molecular masses of 6 to 54 kDa. ¹⁹F labeling is more promising than ¹⁵N, ¹³C, and ²H enrichment. We also used ¹⁹F NMR spectroscopy to quantify the contribution of viscosity, weak interactions, and sample inhomogeneity to resonance broadening in cells. We found that the viscosity in oocytes is only about 1.2 times that of water, and that inhomogeneous broadening is a major factor in determining line width in these cells.     

Protein Structure Determination with Three- and Four-Dimensional NMR Spectroscopy

Structural biology has made important contributions to the understanding of biological processes. In recent years an increasing amount of structural information has also been derived from NMR spectroscopic studies, often with special emphasis on dynamic aspects. The introduction of three- and four-dimensional techniques has greatly simplified protein structure determination by NMR Spectroscopy, which has in fact become routine. In the past it was more of an art to interpret the complicated NOESY spectra of proteins, but the application of three-dimensional techniques now makes the interpretation of protein spectra straightforward. In this review we discuss the most important multidimensional NMR techniques along with suitable applications. The emphasis is put less on the discussion of individual pulse sequences than on their application to the structure determination of proteins.     

Perylene Bisimide Dimer Aggregates: Fundamental Insights into Self‐Assembly by NMR and UV/Vis Spectroscopy

A novel perylene bisimide (PBI) dye bearing one solubilizing dialkoxybenzyl and one bulky 2,5‐di‐tert‐butylphenyl substituent was synthesized and its aggregation behavior was analyzed by NMR and UV/Vis spectroscopy in various chloroform/methylcyclohexane (MCH) solvent mixtures. In the presence of no less than 10 vol % chloroform, exclusive self‐assembly of this PBI dye into π‐stacked dimers was unambiguously confirmed by means of both concentration‐dependent ¹H NMR and UV/Vis spectroscopic experiments. Based on ROESY NMR, a well‐defined π‐stacked dimer structure was determined and further corroborated by molecular modeling studies. By varying the solvent composition of chloroform and MCH, the solvent effects on the Gibbs free energy of PBI dimerization were elucidated and showed a pronounced nonlinearity between lower and higher MCH contents. This observation could be related to a further growth process of dimers into larger aggregates that occurs in the absence of chloroform, which is required to solvate the aromatic π surfaces. With the help of a single‐crystal structure analysis for a related PBI dye, a structural model could be derived for the extended aggregates that are still composed of defined π–π‐stacked PBI dimer entities.