Two-dimensional J-resolved NMR Spectroscopy Research Articles

Observation of the Imidazole-Imidazolium Hydrogen Bonds Responsible for Selective Proton Conductance in the Influenza A M2 Channel.

The integral membrane M2 protein is a 97-residue membrane protein that assembles as a tetramer to conduct protons at a slow rate (102-103/s) when activated by low pH. The proton conductance mechanism has been extensively debated in the literature, but it is accepted that the proton conductance is facilitated by hydrogen bonds involving the His37 residues. However, the hydrogen bonding partnership remains unresolved. Here, we report on the measurement of 15N-15N J-couplings of 15N His37-labeled full length M2 (M2FL) protein from Influenza A virus embedded in synthetic liquid crystalline lipid bilayers using two-dimensional J-resolved NMR spectroscopy. We experimentally observed the hydrogen-bond mediated J-couplings between Nδ1 and Nε2 of adjacent His37 imidazole rings, providing direct evidence for the existence of various imidazolium-imidazole hydrogen-bonding geometries in the histidine tetrad at low pH, thus validating the proton conduction mechanism in the M2FL protein by which the proton is transferred through the breaking and reforming of the hydrogen bonds between pairs of His37 residues.

Spatially Localized Two-Dimensional J-Resolved NMR Spectroscopy via Intermolecular Double-Quantum Coherences for Biological Samples at 7 T.

Background and PurposeMagnetic resonance spectroscopy (MRS) constitutes a mainstream technique for characterizing biological samples. Benefiting from the separation of chemical shifts and J couplings, spatially localized two-dimensional (2D) J-resolved spectroscopy (JPRESS) shows better identification of complex metabolite resonances than one-dimensional MRS does and facilitates the extraction of J coupling information. However, due to variations of macroscopic magnetic susceptibility in biological samples, conventional JPRESS spectra generally suffer from the influence of field inhomogeneity. In this paper, we investigated the implementation of the localized 2D J-resolved spectroscopy based on intermolecular double-quantum coherences (iDQCs) on a 7 T MRI scanner.Materials and MethodsA γ-aminobutyric acid (GABA) aqueous solution, an intact pig brain tissue, and a whole fish (Harpadon nehereus) were explored by using the localized iDQC J-resolved spectroscopy (iDQCJRES) method, and the results were compared to those obtained by using the conventional 2D JPRESS method.ResultsInhomogeneous line broadening, caused by the variations of macroscopic magnetic susceptibility in the detected biological samples (the intact pig brain tissue and the whole fish), degrades the quality of 2D JPRESS spectra, particularly when a large voxel is selected and some strongly structured components are included (such as the fish spinal cord). By contrast, high-resolution 2D J-resolved information satisfactory for metabolite analyses can be obtained from localized 2D iDQCJRES spectra without voxel size limitation and field shimming. From the contrastive experiments, it is obvious that the spectral information observed in the localized iDQCJRES spectra acquired from large voxels without field shimming procedure (i.e. in inhomogeneous fields) is similar to that provided by the JPRESS spectra acquired from small voxels after field shimming procedure (i.e. in relatively homogeneous fields).ConclusionThe localized iDQCJRES method holds advantage for recovering high-resolution 2D J-resolved information from inhomogeneous fields caused by external non-ideal field condition or internal macroscopic magnetic susceptibility variations in biological samples, and it is free of voxel size limitation and time-consuming field shimming procedure. This method presents a complementary way to the conventional JPRESS method for MRS measurements on MRI systems equipped with broad inner bores, and may provide a promising tool for in vivo MRS applications.

High-Resolution Two-Dimensional J-Resolved NMR Spectroscopy for Biological Systems

NMR spectroscopy is a principal tool in metabolomic studies and can, in theory, yield atom-level information critical for understanding biological systems. Nevertheless, NMR investigations on biological tissues generally have to contend with field inhomogeneities originating from variations in macroscopic magnetic susceptibility; these field inhomogeneities broaden spectral lines and thereby obscure metabolite signals. The congestion in one-dimensional NMR spectra of biological tissues often leads to ambiguities in metabolite identification and quantification. We propose an NMR approach based on intermolecular double-quantum coherences to recover high-resolution two-dimensional (2D) J-resolved spectra from inhomogeneous magnetic fields, such as those created by susceptibility variations in intact biological tissues. The proposed method makes it possible to acquire high-resolution 2D J-resolved spectra on intact biological samples without recourse to time-consuming shimming procedures or the use of specialized hardware, such as magic-angle-spinning probes. Separation of chemical shifts and J couplings along two distinct dimensions is achieved, which reduces spectral crowding and increases metabolite specificity. Moreover, the apparent J coupling constants observed are magnified by a factor of 3, facilitating the accurate measurement of small J couplings, which is useful in metabolic analyses. Dramatically improved spectral resolution is demonstrated in our applications of the technique on pig brain tissues. The resulting spectra contain a wealth of chemical shift and J-coupling information that is invaluable for metabolite analyses. A spatially localized experiment applied on an intact fish (Crossocheilus siamensis) reveals the promise of the proposed method in in vivo metabolite studies. Moreover, the proposed method makes few demands on spectrometer hardware and therefore constitutes a convenient and effective manner for metabonomics study of biological systems.

Structural interpretation of vicinal proton-proton coupling constants 3JH alpha H beta in the basic pancreatic trypsin inhibitor measured by two-dimensional J-resolved NMR spectroscopy.

Two-dimensional J-resolved 1H NMR spectroscopy was used to measure the vicinal spin-spin coupling constants 3JH alpha H beta for numerous, previously individually assigned amino acid residues in the basic pancreatic trypsin inhibitor at various temperatures between 30 and 85 degrees JC. An analysis of this data is proposed which enables one to compare the spatial arrangements of individual amino acid side chains in solution and in single crystals of the protein, and which also provides information on the mobility of the side chains in the solution conformation. As a rule, the amino acid side chains in the interior of the protein were found to be locked into unique spatial orientations, with the mobility restricted to rapid rotational fluctuations about this unique value for the dihedral angle chi 1. In most, but not all, instances the data for the interior amino acids indicate identical average conformations for the amino acid side chains in single crystals and in solution. For residues on the protein surface structural rearrangements between crystal and solution appear to be common, and the mobility in the solution conformation may include rapid averaging between two or several distinct, preferentially populated values of chi 1, analogous to the gauche-trans-gauche isomerization in isolated amino acids.

Absolute configurational assignments for the1H-NMR spectrum of poly(vinyl alcohol) by use of two-dimensional NMR methods

FOCSY two-dimensional J-resolved NMR spectroscopy is used to resolve1H NMR resonances corresponding to triad and tetrad configurations of poly(vinyl alcohol). Two-dimensional auto correlation with ω1-scaling NMR spectroscopy is used to observe connectivities between triad and tetrad1H NMR resonances. Comparison of these connectivities to the necessary compositional relationships at triad-tetrad level leads to absolute configurational assignments for the1H NMR spectrum of poly(vinyl alcohol).

Two-dimensional J-resolved proton NMR spectroscopy of oligomannosidic glycopeptides.

Individual anomeric protons that are unresolved in the one-dimensional 250-MHz spectra of oligomannosidic glycopeptides can be separated and characterized by two-dimensional J-resolved NMR spectroscopy. Homogeneous preparations of ovalbumin glycopeptides Man6GlcNAc2Asn and Man5GlcNAc2Asn were characterized by chemical methods, conventional proton NMR, and two-dimensional J-resolved NMR. Due to characteristic differences in coupling constants, mannose (J1,2 less than or equal to 2 Hz) and N-acetylglucosamine (J1,2 approximately 9 Hz) anomeric signals of similar chemical shift were readily separated and identified in the two-dimensional spectra. It is shown that two-dimensional J-resolved NMR spectroscopy, in combination with conventional NMR and limited chemical analysis, is a rapid and reliable technique for the determination of glycopeptide primary structure.

Spin decoupling in two-dimensional J-resolved NMR spectroscopy

Spin decoupling techniques are described which are suitable to enhance the utility of two-dimensional J-resolved NMR spectroscopy with regard to (a) simplification of 2D spectra, and (b) assignment of resonance peaks. The theoretical foundations are developed to analyze three different types of double resonance experiments, which differ by the time period during which decoupling is applied. The theoretical predictions are confirmed by experiments on the AX spin system of 2,3,4-trichlorophenol. An extension of this technique permits the recording of pure phase 2D J-resolved NMR spectra.