High-resolution Nuclear Magnetic Resonance Studies Research Articles

The early days of ex vivo 1 H, 13 C, and 31 P nuclear magnetic resonance in the laboratory of Dr. Robert G. Shulman from 1975 to 1995.

This paper provides a brief description of the early use of ex vivo nuclear magnetic resonance (NMR) studies of tissue and tissue extracts performed in the laboratory of Dr. Robert G. Shulman from 1975 through 1995 at Bell Laboratories, then later at Yale University. During that period, ex vivo NMR provided critical information in support of resonance assignments and the quantitation of concentrations for magnetic resonance spectroscopy studies. The period covered saw rapid advances in magnet technology, starting with studies of microorganisms in vertical bore high-resolution NMR studies, then by 1981 studies of small mammals in a horizontal bore magnet, and then studies of humans in 1984. Ex vivo NMR played a critical role in all these studies. A general strategy developed in the lab for using ex vivo NMR to support in vivo studies is presented, as well as illustrative examples.

Solid-state NMR analysis of unlabeled fungal cell walls from Aspergillus and Candida species

Fungal infections cause high mortality in immunocompromised individuals, which has emerged as a significant threat to human health. The efforts devoted to the development of antifungal agents targeting the cell wall polysaccharides have been hindered by our incomplete picture of the assembly and remodeling of fungal cell walls. High-resolution solid-state nuclear magnetic resonance (ss NMR) studies have substantially revised our understanding of the polymorphic structure of polysaccharides and the nanoscale organization of cell walls in Aspergillus fumigatus and multiple other fungi. However, this approach requires 13C/15N-enrichment of the sample being studied, severely restricting its application. Here we employ the dynamic nuclear polarization (DNP) technique to compare the unlabeled cell wall materials of A. fumigatus and C. albicans prepared using both liquid and solid media. For each fungus, we have identified a highly conserved carbohydrate core for the cell walls of conidia and mycelia, and from liquid and solid cultures. Using samples prepared in different media, the recently identified function of α-glucan, which packs with chitin to form the mechanical centers, has been confirmed through conventional ss NMR measurements of polymer dynamics. These timely efforts not only validate the structural principles recently discovered for A. fumigatus cell walls in different morphological stages, but also open up the possibility of extending the current investigation to other fungal materials and cellular systems that are challenging to label.

Nuclear Magnetic Resonance-Based Structural Characterization and Backbone Dynamics of Recombinant Bee Venom Melittin.

In recent years, there has been a resurgence of interest in melittin and its variants as their therapeutic potential has become increasingly evident. Melittin is a 26-residue peptide and a toxic component of honey bee venom. The versatility of melittin in interacting with various biological substrates, such as membranes, glycosaminoglycans, and a variety of proteins, has inspired a slew of studies that aim to improve our understanding of the structural basis of such interactions. However, these studies have largely focused on melittin solutions at high concentrations (>1 mM), even though melittin is generally effective at lower (micromolar) concentrations. Here we present high-resolution nuclear magnetic resonance studies in the lower-concentration regime using a novel method to produce isotope-labeled (15N and 13C) recombinant melittin. We provide residue-specific structural characterization of melittin in dilute aqueous solution and in 2,2,2-trifluoroethanol/water mixtures, which mimic melittin structure-function and interactions in aqueous and membrane-like environments, respectively. We find that the cis-trans isomerization of Pro14 is key to changes in the secondary structure of melittin. Thus, this study provides residue-specific structural information about melittin in the free state and in a model of the substrate-bound state. These results, taken together with published work from other laboratories, reveal the peptide's structural versatility that resembles that of intrinsically disordered proteins and peptides.

Understanding the Effect of Disease-Related Mutations on Human Prion Protein Structure: Insights From NMR Spectroscopy.

Prion diseases or transmissible spongiform encephalopathies constitute a group of fatal neurodegenerative diseases that can be of sporadic, genetic, or acquired origin. The central molecular event of prion diseases is the conformational conversion of the physiological cellular prion protein, PrPC, into a disease-associated form known as prion or PrPSc. Spontaneous generation of prions in genetic prion diseases is caused by mutations in the human prion protein gene (PRNP). Understanding of the earliest conformational changes during misfolding of PrPC in genetic forms of prion diseases may benefit from detailed structural characterization of various human (Hu) PrP variants. Nuclear magnetic resonance (NMR) spectroscopy offers unique opportunities to obtain detailed atomic-level structure information. In this chapter we present an overview of high-resolution NMR studies on several HuPrPs with disease-associated mutations at mildly acidic and physiological pH conditions that provided valuable insights into possible key structural determinants underlying the formation of prions.

High resolution NMR study of T1 magnetic relaxation dispersion. IV. Proton relaxation in amino acids and Met-enkephalin pentapeptide

Nuclear Magnetic Relaxation Dispersion (NMRD) of protons was studied in the pentapeptide Met-enkephalin and the amino acids, which constitute it. Experiments were run by using high-resolution Nuclear Magnetic Resonance (NMR) in combination with fast field-cycling, thus enabling measuring NMRD curves for all individual protons. As in earlier works, Papers I-III, pronounced effects of intramolecular scalar spin-spin interactions, J-couplings, on spin relaxation were found. Notably, at low fields J-couplings tend to equalize the apparent relaxation rates within networks of coupled protons. In Met-enkephalin, in contrast to the free amino acids, there is a sharp increase in the proton T1-relaxation times at high fields due to the changes in the regime of molecular motion. The experimental data are in good agreement with theory. From modelling the relaxation experiments we were able to determine motional correlation times of different residues in Met-enkephalin with atomic resolution. This allows us to draw conclusions about preferential conformation of the pentapeptide in solution, which is also in agreement with data from two-dimensional NMR experiments (rotating frame Overhauser effect spectroscopy). Altogether, our study demonstrates that high-resolution NMR studies of magnetic field-dependent relaxation allow one to probe molecular mobility in biomolecules with atomic resolution.

High-resolution 2D NMR spectra in inhomogeneous fields via 3D acquisition

High-resolution nuclear magnetic resonance (NMR) spectroscopy plays an important role in chemical studies. Here, a pulse sequence, based on coherence transfer module of tracking differences of precession frequencies of two spins and spin echo module, is proposed to obtain two dimension (2D) high-resolution NMR spectra via 3D acquisition under large field inhomogeneity. The proposed scheme composes of simple hard pulses and rectangle gradients. Resulting 2D spectra exhibit chemical shift differences and J coupling splittings in two orthogonal dimensions. The method developed here may offer a promising way for in situ high-resolution NMR studies on combinatorial chemistry.

ChemInform Abstract: High-Resolution Solid-State NMR Studies of Synthetic and Biological Macromolecules

Publisher Summary This chapter reviews the high-resolution solid-state nuclear magnetic resonance (NMR) studies of synthetic polymers and naturally occurring biological macromolecules with emphasis placed on revealing their conformations. A survey of high-resolution solid-state NMR studies of both types of macromolecular system has the advantage of being able to cover the topics of conformation and dynamics of polymers in general. A proportion of the studies reviewed have been devoted to the analysis of secondary structures in biological macromolecules, such as peptides, proteins, and polysaccharides, while the majority of the studies reviewed have been performed for the analysis of phase structure, the various modes of molecular motions, and their influence on the mechanical properties of synthetic polymers. The scope of this chapter is restricted mainly to studies of 13C nuclei, although some studies using other rare spin 1/2 nuclei, such as 31Po r 15N are reviewed where appropriate. This is because the use of 13C NMR is most suitable for organic macromolecules and the majority of studies on these subjects have been caried out by the means of 13C NMR spectroscopy.

Peripherin-2: An Intracellular Analogy to Viral Fusion Proteins

The C-terminus of the intracellular retinal rod outer segment disk protein peripherin-2 binds to membranes, adopts a helical conformation, and promotes membrane fusion, which suggests an analogy to the structure and function of viral envelope fusion proteins. Nuclear magnetic resonance (NMR) data and fluorescence data show that a 63-residue polypeptide comprising the C-terminus of bovine peripherin-2 (R284-G346) binds to the membrane mimetic, dodecylphosphocholine micelles. High-resolution NMR studies reveal that although this C-terminal fragment is unstructured in solution, the same fragment adopts helical structure when bound to the micelles. The C-terminus may be a member of the class of intrinsically unstructured protein domains. Using methods developed for the G-protein coupled receptor rhodopsin, a model for the structure of the transmembrane domain of peripherin-2 was constructed. Previously published data showed that both peripherin-2 and viral fusion proteins are transmembrane proteins that promote membrane fusion and have a fusion peptide sequence within the protein that independently promotes membrane fusion. Furthermore, the fusion-active sequence of peripherin-2 exhibits a sequence motif that matches the viral fusion peptide of influenza hemagglutinin (HA). These observations collectively suggest that the mechanism of intracellular membrane fusion induced by peripherin-2 and the mechanism of enveloped viral fusion may have features in common.

The Application of NMR-Based Metabonomics in Neurological Disorders

Advances in postgenomic technologies have radically changed the information output from complex biological systems, generating vast amounts of high complexity data that can be interpreted by means of chemometric and bioinformatic methods to achieve disease diagnosis and prognosis. High-resolution nuclear magnetic resonance (NMR) spectroscopy of biofluids such as plasma, cerebrospinal fluid (CSF), and urine can generate robust, interpretable metabolic fingerprints that contain latent information relating to physiological or pathological status. This technology has been successfully applied to both preclinical and clinical studies of neurodegenerative diseases such as Huntington's disease, muscular dystrophy, and cerebellar ataxia. An extension of this technology, (1)H magic-angle-spinning (HRMAS) NMR spectroscopy, can be used to generate metabolic information on small intact tissue samples, providing a metabolic link between metabolic profiling of biofluids and histology. In this review we provide a summary of high-resolution NMR studies in neurodegenerative disease and explore the potential of metabonomics in evaluating disease progression with respect to therapeutic intervention.

Thermodynamic and structural factors in the removal of bulky DNA adducts by the nucleotide excision repair machinery.

The function of the human nucleotide excision repair (NER) apparatus is to remove bulky adducts from damaged DNA. In an effort to gain insights into the molecular mechanisms involved in the recognition and excision of bulky lesions, we investigated a series of site specifically modified oligonucleotides containing single, well-defined polycyclic aromatic hydrocarbon (PAH) diol epoxide-adenine adducts. Covalent adducts derived from the bay region PAH, benzo[a]pyrene, are removed by human NER enzymes in vitro. In contrast, the stereochemically analogous N(6)-dA adducts derived from the topologically different fjord region PAH, benzo[c]phenanthrene, are resistant to repair. The evasion of DNA repair may play a role in the observed higher tumorigenicity of the fjord region PAH diol epoxides. We are elucidating the structural and thermodynamic features of these adducts that may underlie their marked distinction in biologic function, employing high-resolution nuclear magnetic resonance studies, measurements of thermal stabilities of the PAH diol epoxide-modified oligonucleotide duplexes, and molecular dynamics simulations with free energy calculations. Our combined findings suggest that differences in the thermodynamic properties and thermal stabilities are associated with differences in distortions to the DNA induced by the lesions. These structural effects correlate with the differential NER susceptibilities and stem from the intrinsically distinct shapes of the fjord and bay region PAH diol epoxide-N(6)-adenine adducts.

Effective Computational Strategies for Determining Structures of Carcinogen-Damaged DNA

To determine three-dimensional conformations of DNA damaged by environmental chemical carcinogens, effective molecular mechanics search techniques have been developed to deal with the large system sizes and computational demands. First, extensive surveys of the potential energy surface are carried out by energy minimization. These search strategies rely on (1) using the reduced variable domain of torsion-angle (rather than Cartesian) space, (2) building larger units (about 12 base pairs) on the basis of structures of small modified subunits, and (3) employing penalty functions to search for selected hydrogen bonding patterns and to incorporate interproton distance bounds when available from experimental high-resolution nuclear magnetic resonance (NMR) studies. Second, molecular dynamics simulations with solvent can subsequently be employed to probe conformational features in the presence of polymerase enzyme responsible for DNA replication, using structures computed in the energy minimization searches as initial coordinates. A key structure–function relationship involving mirror-image molecules with very differing experimentally determined tumorigenic potencies has been deduced: the members of the pairs align oppositely when bound to DNA, making it likely that their treatment by replication and repair enzymes differ. This opposite orientation phenomenon, first predicted computationally (Singhet al., 1991), has been observed in experimental high-resolution NMR studies combined with our molecular mechanics computations in a number of different examples and has recently been confirmed experimentally in other laboratories as well (reviewed in Geacintovet al., 1997). Elucidation of this conformational feature has paved the way to uncovering the structural origin underlying very different biological outcomes stemming from chemically identical but mirror-image molecules.

High-resolution NMR studies of nifedipine

The paper reports results of1H and13C high-resolution nuclear magnetic resonance studies of nifedipine and signal assignment to particular atoms. High-resolution1H NMR spectra with no signals attributed to protons in weak fields (δ= 10−12 ppm) imply that H7 atom is strongly connected to the ring and is not involved in the formation of hydrogen bonds.

High-resolution magic-angle spinning 1H nuclear magnetic resonance studies of lipid dispersions using spherical glass ampoules

A very useful high-resolution magic-angle spinning (MAS) 1H NMR method for studying lipid dispersions is presented. The sample can be loaded into the spherical glass ampoule very easily, and a spinning speed of more than 10 kHz can be achieved without the problems of sample leakage or water loss. The line width at half height for the HDO peak is less than 1.5 Hz, and the method can be implemented by anyone who has access to a solid-state MAS NMR spectrometer. By using the spherical glass ampoule method we found two water peaks, which could be ascribed to bulk water outside of the multilamellar liposome (peak at high frequency) and interlamellar water (peak at low frequency), in both POPC (1-palmitoyl-2-oleoyl- sn-glycero-3-phosphocholine) and DOPC (1,2-dioleoyl- sn-glycero-3-phosphocholine) liposomes. These are the first two examples of lipids without exchangeable protons that exhibit two distinct water resonances. The respective 1H spin-lattice relaxation times ( T 1) were measured, yielding values twice as long for bulk water as compared with interlamellar water. Both the chemical shift and spin relaxation results demonstrate the ability of MAS 1H NMR to rapidly monitor changes in physical properties that accompany water interactions with zwitterionic phosphatidylcholines.

High-resolution solid-state magic-angle spinning nuclear magnetic resonance studies on the layered antimony hydrogen phosphate, HSb(PO 4) 2·2H 2O and some organic amine intercalation compounds

n-propylamine and tri- n-octylamine were intercalated into the layered host antimony hydrogen phosphate, HSb(PO 4) 2·2H 2O (henceforth written as H 1SbP 2). Powder X-ray diffraction (PXRD) indicated that the reaction products were all polyphasic microcrystalline materials and suggested that the arrangement of amine molecules in (i) the n-propylamine intercalation product were similar to that observed for amine intercalated α-Zr(HPO 4) 2 and α-tin (IV) hydrogen phosphates, and that (ii) the tri- n-octylamine molecules formed a bilayered structure within the H 1SbP 2 interlayer in which the terminal methyl groups repulse one another. Fourier transformed infrared (FTIR) studies also suggested the presence of intercalated amine molecules within the H 1SbP 2 layered structure. High-resolution 15 N solid-state magic-angle spinning nuclear magnetic resonance (MAS NMR) indicated that for both reaction products a single amine species was intercalated into the H 1SbP 2 interlamellar region as expected. 31 P solid-state MAS NMR studies confirmed that the intercalating amine species were all protonated and that in the case of the n-propylamine reaction products, there were a variety of orientated intercalating species. 31 P MAS NMR also suggested that there was perturbation of the adjacent phosphate groups by the large tri- n-octylamine alkyl chains. This type of perturbation has previously been observed for iron complexes intercalated into the H 1SbP 2 host.

High-resolution solid-state magic angle spinning nuclear magnetic resonance studies on the layered antimony hydrogen phosphate, HSb(PO4)2·2H2O, and its reaction products with tetrakis(pyridyl)iron(II) chloride

The antimony hydrogen phosphate, HSb(PO 4 ) 2 ·2H 2 O (denoted H 1 SbP 2 ·2H 2 O), has been synthesised via the ion exchange of crystalline KSb(PO 4 ) 2 , (K 1 SbP 2 ), with 9 m HNO 3 . Scanning electron microscopy (SEM) confirmed that the crystalline K 1 SbP 2 was formed by the so-called ‘deck of cards’ mechanism to give randomly orientated lamellae. The synthesised H 1 SbP 2 ·2H 2 O host material was studied using 1 H and 31 P magic angle spinning nuclear magnetic resonance (MAS NMR) techniques. Intercalation studies were carried out using tetrakis(pyridyl)iron(ii) chloride, [Fe(py) 4 Cl 2 ]·H 2 O. The resulting products were analysed using powder X-ray diffraction (PXRD) and 31 P MAS NMR techniques. The former suggested that the [Fe(py) 4 Cl 2 ]·H 2 O complex lost its water of crystallisation during the reaction and did not intercalate in its intact state between adjacent layers of the H 1 SbP 2 ·2H 2 O crystallites. 31 P MAS NMR data suggested that the H 1 SbP 2 ·2H 2 O–[Fe(py) 4 Cl 2 ] reaction products contained phosphorus resonances which could be assigned as belonging to (i) unaltered host H 1 SbP 2 ·2H 2 O (protonated) phosphate groups, (ii) phosphate groups bonded to the intercalating species. In addition, a separate, Q P 3 , resonance was also noted which was thought to arise from a chemically unaltered phosphate group of the host H 1 SbP 2 ·2H 2 O perturbed by the close proximity of the sorbed [Fe(py) 4 ] 2+ cationic species.

High-resolution proton nuclear magnetic resonance studies of urine from asphyxiated newborn infants

High-resolution proton nuclear magnetic resonance spectroscopy was used to study human urine obtained from 10 normal babies and twenty babies with various degrees of neonatal asphyxia, respiratory distress syndrome (RDS), and meconium aspiration syndrome (MAS). All sick babies showed different degrees of oxygen deficiency, indicated by an obvious increase of the lactate signal level in the urine spectra. Changes in the concentration of other urinary metabolites produced from the citric acid cycle were also observed. In extremely serious cases, the signals of some of the major components, including citrate, alpha-ketoglutarate, and succinate, simply disappeared. The spectra of urine, serum, and CSF of an infant suffering from SIDS showed common characteristics of the metabolites.

1H and 51V high-resolution solid state nuclear magnetic resonance studies of supported V 2O 5/TiO 2 catalysts

1H solid state NMR data show the existence of several types of hydroxyl groups on TiO 2 surface, depending on the surface impurities. VOCl 3 selectively interacts with these groups. According to 51V NMR data, the structure of the surface vanadium complexes, independently of the preparation method, is determined by the type of surface hydroxyls. On clean TiO 2 surface at low vanadium concentration two types of surface complexes with vanadium in the distorted tetrahedral environment of oxygen atoms are formed, one of them containing OH groups in the coordination sphere. At high V content associated species, with V in octahedral coordination, are formed. nuclear magnetic resonance studies; solid state; titania; vanadia

Cloning, expression, and crystallization of recoverin, a calcium sensor in vision.

Recoverin, a recently discovered 23-kDa calcium-binding protein, activates retinal rod guanylate cyclase when the calcium level is lowered in the submicromolar range. We report here the cloning and sequencing of a cDNA for recoverin from a bovine retinal expression library. The recoverin coding sequence was inserted into a pET-11a expression vector under control of the T7 phage promoter. A second expression system, in which the coding sequence was placed under control of the lambda phage PR promoter, gave 10-fold higher yields (10 mg of purified recoverin per liter of Escherichia coli culture). The finding that retinal recoverin is myristoylated at its amino terminus led us to coexpress the recombinant protein and N-myristoyltransferase (EC 2.3.1.97). Myristoylated recombinant recoverin formed in this way in E. coli is like retinal recoverin in exhibiting a large calcium-induced shift in its tryptophan fluorescence emission spectrum. The availability of abundant protein enabled us to crystallize unmyristoylated recombinant recoverin and initiate x-ray studies. The space group of tetragonal crystals obtained from 75% saturation ammonium sulfate is I4 with unit cell dimensions a = 85.1 A and c = 59.8 A. These crystals of the calcium-bound form of the protein diffracted to a resolution of 2.2 A. The expression systems described here open the door to high-resolution x-ray crystallographic and nuclear magnetic resonance studies of this new member of the EF-hand superfamily and to the elucidation of its precise mode of action as a calcium switch.

1 3C high resolution nuclear magnetic resonance studies in polycrystalline tetracyanoquinodimethane

1 3C high resolution NMR (nuclear magnetic resonance) studies of solid neutral tetracyanoquinodimethane are performed using different polycrystalline samples: (i) natural isotopic abundance; (ii) 15N isotope enriched; (iii) 13C isotope selectively enriched on the CN groups; and (iv) 13C isotope selectively enriched on the first neighboring carbons of the CN groups. 14N quadrupole effects are observed on 13C spectra of the CN groups, and the quadrupole coupling constant for 14N is obtained: −3.84±0.12 MHz. The 13C isotropic chemical shifts are found: 112.6 ppm for CN groups; 90.8 ppm for the carbons directly bonded to these groups; 132.0 and 133.2 ppm for protonated carbons; and 151.7 ppm for the other carbons on the quinone ring. The principal components of the chemical shielding tensor, the shielding anisotropy, and the shielding asymmetry factor are also obtained for the 13C nuclei in the fragments C–(CN)2, by graphical analysis of CP/MAS spectra. The following values are obtained for 13C nuclei on CN groups: 211±11 ppm, −85±22 ppm (σ⊥ and σ∥); −296±33 ppm (Δσ), and 0 (η). For the other carbon nuclei the values obtained for σ11, σ22, σ33, Δσ, and η are: 157±2 ppm, 75±2 ppm, 39±3 ppm, −77±4 ppm and 1.6±0.1, respectively. The reliability of these results is confirmed by a computer fit of the line intensities.

Protein Hydration in Aqueous Solution

High-resolution proton nuclear magnetic resonance studies of protein hydration in aqueous solution show that there are two qualitatively different types of hydration sites. A well-defined, small number of water molecules in the interior of the protein are in identical locations in the crystal structure and in solution, and their residence times are in the range from about 10(-2) to 10(-8) second. Hydration of the protein surface in solution is by water molecules with residence times in the subnanosecond range, even when they are located in hydration sites that contain well-ordered water in the x-ray structures of protein single crystals.