Project description:The typical linewidths of 1 H NMR spectra of powdered organic solids at 111 kHz magic-angle spinning (MAS) are of the order of a few hundred Hz. While this is remarkable in comparison to the tens of kHz observed in spectra of static samples, it is still the key limit to the use of 1 H in solid-state NMR, especially for complex systems. Here, we demonstrate a novel strategy to further improve the spectral resolution. We show that the anti-z-COSY experiment can be used to reduce the residual line broadening of 1 H NMR spectra of powdered organic solids. Results obtained with the anti-z-COSY sequence at 100 kHz MAS on thymol, β-AspAla, and strychnine show an improvement in resolution of up to a factor of two compared to conventional spectra acquired at the same spinning rate.
Project description:The Bloch-Siegert shift is a phenomenon in NMR spectroscopy and atomic physics in which the observed resonance frequency is changed by the presence of an off-resonance applied field. In NMR, it occurs especially in the context of homonuclear decoupling. Here we develop a practical method for homonuclear decoupling that avoids inducing Bloch-Siegert shifts. This approach enables accurate observation of the resonance frequencies of decoupled nuclear spins. We apply this method to increase the resolution of the HNCA experiment. We also observe a doubling in sensitivity for a 30?kDa protein. We demonstrate the use of band-selective C? decoupling to produce amino acid-specific line shapes, which are valuable for assigning resonances to the protein sequence. Finally, we assign the backbone of a 30?kDa protein, Human Carbonic Anhydrase II, using only HNCA experiments acquired with band-selective decoupling schemes, and instrument time of one week.
Project description:Over the years, NMR spectroscopy has become a powerful analytical tool for the identification and quantification of a variety of natural compounds in a broad range of food matrices. Furthermore, NMR can be useful for characterizing food matrices in terms of quality and authenticity, also allowing for the identification of counterfeits. Although NMR requires minimal sample preparation, this technique suffers from low intrinsic sensitivity relative to complementary techniques; thus, the detection of adulterants or markers for authenticity at low concentrations remains challenging. Here, we present a strategy to overcome this limitation by the introduction of a simple band-selective homonuclear decoupling sequence that consists of double irradiation on 1H during NMR signal acquisition. The utility of the proposed method is tested on dihydrosterculic acid (DHSA), one of the cyclopropane fatty acids (CPFAs) shown to be a powerful molecular marker for authentication of milk products. A quantitative description of how the proposed NMR scheme allows sensitivity enhancement yet accurate quantification of DHSA is provided.
Project description:The 1 H NMR analysis of species containing NMR-active heteronuclei can be difficult due to signal overlap caused by the combined effects of homonuclear and heteronuclear scalar (J) couplings. Here, a general pure shift method is presented for obtaining ultra-high resolution 1 H NMR spectra where spectral overlap is drastically reduced by suppressing both homonuclear and heteronuclear J-couplings, giving one single signal per 1 H chemical environment. Its usefulness is demonstrated in the analysis of fluorine- and phosphorus-containing compounds of pharmaceutical and biochemical interest.
Project description:Sensitivity and resolution are the two fundamental obstacles to extending solid-state nuclear magnetic resonance to even larger protein systems. Here, a novel long-observation-window band-selective homonuclear decoupling (LOW BASHD) scheme is introduced that increases resolution up to a factor of 3 and sensitivity up to 1.8 by decoupling backbone alpha-carbon (C(α)) and carbonyl (C') nuclei in U-(13)C-labeled proteins during direct (13)C acquisition. This approach introduces short (<200 μs) pulse breaks into much longer (~8 ms) sampling windows to efficiently refocus the J-coupling interaction during detection while avoiding the deleterious effects on sensitivity inherent in rapid stroboscopic band-selective homonuclear decoupling techniques. A significant advantage of LOW-BASHD detection is that it can be directly incorporated into existing correlation methods, as illustrated here for 2D CACO, NCO, and NCA correlation spectroscopy applied to the β1 immunoglobulin binding domain of protein G and 3D CBCACO correlation spectroscopy applied to the α-subunit of tryptophan synthase.
Project description:2D homonuclear NMR spectroscopy is an essential technique to characterize small and large molecules, such as organic compounds, metabolites, and biomacromolecules at atomic resolution. However, for complex samples 2D homonuclear spectra display poor resolution, making spectral assignment very cumbersome. Here, we propose a new method that exploits the differential T2* relaxation times of individual resonances and resolves the 2D NMR peaks into pseudo-3D spectra, where time is the 3rd dimension. T2* weIghted DEconvolution or TIDE analyzes individual free induction decays (FIDs) and dissects them into sub-FIDs that are transformed into pseudo-3D spectra combining Fourier transformation and covariance NMR. TIDE achieves higher resolution and sensitivity for NMR spectra than classical covariance NMR reducing offset-dependent artifacts. We demonstrate the performance of TIDE for magic angle spinning (MAS) [13C,13C]-DARR NMR spectra of single- and multi-span membrane proteins embedded in lipid bilayers. Since TIDE is applicable to all type of homonuclear correlation experiments for liquid and solid samples, we anticipate that it will be a general method for processing NMR data of biomacromolecules, complex mixtures of metabolites as well as material samples.
Project description:Modern applications of 2D NMR spectroscopy to diagnostic screening, metabolomics, quality control, and other high-throughput applications are often limited by the time-consuming sampling requirements along the indirect time domain t1 . 2D total correlation spectroscopy (TOCSY) provides unique spin connectivity information for the analysis of a large number of compounds in complex mixtures, but standard methods typically require >100 t1 increments for an accurate spectral reconstruction, rendering these experiments ineffective for high-throughput applications. For a complex metabolite mixture it is demonstrated that absolute minimal sampling (AMS), based on direct fitting of resonance frequencies and amplitudes in the time domain, yields an accurate spectral reconstruction of TOCSY spectra using as few as 16 t1 points. This permits the rapid collection of homonuclear 2D NMR experiments at high resolution with measurement times that previously were only the realm of 1D experiments.
Project description:Homonuclear two- and multidimensional NMR spectra are standard experiments for the structure determination of small to medium-sized molecules. In the large majority of homonuclear correlated spectra the diagonal contains the most intense peaks. Cross-peaks near the diagonal could overlap with huge tails of diagonal peaks and can therefore be easily overlooked. Here we present a general method for the suppression of peaks along the diagonal in homonuclear correlated spectra. It is based on a spatially selective excitation followed by the suppression of magnetization which has not changed the frequency during the mixing process. In addition to the auto correlation removal, these experiments are also less affected by magnetic field inhomogeneities due to the slice selective excitation, which on the other side leads to a reduced intensity compared to regular homonuclear correlated spectra.
Project description:Over decades multidimensional NMR spectroscopy has become an indispensable tool for structure elucidation of natural products, peptides and medium sized to large proteins. Heteronuclear single quantum coherence (HSQC) spectroscopy is one of the work horses in that field often used to map structural connectivity between protons and carbons or other hetero nuclei. In overcrowded HSQC spectra, proton multiplet structures of cross peaks set a limit to the power of resolution and make a straightforward assignment difficult. In this work, we provide a solution to improve these penalties by completely removing the proton spin multiplet structure of HSQC cross peaks. Previously reported sideband artefacts are diminished leading to HSQC spectra with singlet responses for all types of proton multiplicities. For sideband suppression, the idea of restricted random delay (RRD) in chunk interrupted data acquisition is introduced and exemplified. The problem of irreducible residual doublet splitting of diastereotopic CH2 groups is simply solved by using a phase sensitive JRES approach in conjunction with echo processing and real time broadband homodecoupling (BBHD) HSQC, applied as a 3D experiment. Advantages and limitations of the method is presented and discussed.
Project description:We recently described a family of experiments for R2n(v) Driven Spin Diffusion (RDSD) spectroscopy suitable for homonuclear correlation experiments under fast MAS conditions [G. Hou, S. Yan, S.J. Sun, Y. Han, I.J. Byeon, J. Ahn, J. Concel, A. Samoson, A.M. Gronenborn, T. Polenova, Spin diffusion drive by R-symmetry sequencs: applications to homonuclear correlation spectroscopy in MAS NMR of biological and organic solids, J. Am. Chem. Soc. 133 (2011) 3943-3953]. In these RDSD experiments, since the broadened second-order rotational resonance conditions are dominated by the radio frequency field strength and the phase shifts, as well as the size of reintroduced dipolar couplings, the different R2n(v) sequences display unique polarization transfer behaviors and different recoupling frequency bandwidths. Herein, we present a series of modified R2n(v) sequences, dubbed COmbined R2n(v)-Driven (CORD), that yield broadband homonuclear dipolar recoupling and give rise to uniform distribution of cross peak intensities across the entire correlation spectrum. We report NMR experiments and numerical simulations demonstrating that these CORD spin diffusion sequences are suitable for broadband recoupling at a wide range of magnetic fields and MAS frequencies, including fast-MAS conditions (?r=40 kHz and above). Since these CORD sequences are largely insensitive to dipolar truncation, they are well suited for the determination of long-range distance constraints, which are indispensable for the structural characterization of a broad range of systems. Using U-(13)C,(15)N-alanine and U-(13)C,(15)N-histidine, we show that under fast-MAS conditions, the CORD sequences display polarization transfer efficiencies within broadband frequency regions that are generally higher than those offered by other existing spin diffusion pulse schemes. A 89-residue U-(13)C,(15)N-dynein light chain (LC8) protein has also been used to demonstrate that the CORD sequences exhibit uniformly high cross peak intensities across the entire chemical shift range.