High resolution MAS-NMR in combinatorial chemistry

High-resolution magic angle spinning (hr-MAS) NMR is a powerful tool for characterizing organic reactions on solid support. Because magic angle spinning reduces the line-broadening due to dipolar coupling and variations in bulk magnetic susceptibility, line widths approaching those obtained in solution-phase NMR can be obtained. The magic angle spinning method is amenable for use in conjunction with a variety of NMR-pulse sequences, making it possible to perform full-structure determinations and conformational analysis on compounds attached to a polymer support. Diffusion-weighted MAS-NMR methods such as SPEEDY (Spin-Echo-Enhanced Diffusion-Filtered Spectroscopy) can be used to remove unwanted signals from the solvent, residual reactants, and the polymer support from the MAS-NMR spectrum, leaving only those signals arising from the resin-bound product. This review will present the applications of high-resolution magic angle spinning NMR for use in combinatorial chemistry research.     

Determination of structural topology of a membrane protein in lipid bilayers using polarization optimized experiments (POE) for static and MAS solid state NMR spectroscopy

The low sensitivity inherent to both the static and magic angle spinning techniques of solid-state NMR (ssNMR) spectroscopy has thus far limited the routine application of multidimensional experiments to determine the structure of membrane proteins in lipid bilayers. Here, we demonstrate the advantage of using a recently developed class of experiments, polarization optimized experiments, for both static and MAS spectroscopy to achieve higher sensitivity and substantial time-savings for 2D and 3D experiments. We used sarcolipin, a single pass membrane protein, reconstituted in oriented bicelles (for oriented ssNMR) and multilamellar vesicles (for MAS ssNMR) as a benchmark. The restraints derived by these experiments are then combined into a hybrid energy function to allow simultaneous determination of structure and topology. The resulting structural ensemble converged to a helical conformation with a backbone RMSD ~0.44 Å, a tilt angle of 24° ± 1°, and an azimuthal angle of 55° ± 6°. This work represents a crucial first step toward obtaining high-resolution structures of large membrane proteins using combined multidimensional oriented solid-state NMR and magic angle spinning solid-state NMR.     

High-resolution solid-state nuclear magnetic resonance spectra of dentin collagen

Insoluble collagen of bovine dentin was characterized by high-resolution solid-state 13C nuclear magnetic resonance (NMR) spectroscopy using a cross-polarization magic angle spinning procedure. A downfield shift was observed in the signal of hydroxyproline C beta compared with that in skin collagen, indicating a distortion in the hydroxyproline structure. A signal of 31P NMR was detected in dentin collagen that was compatible with the presence of matrix-associated phosphoprotein.     

Magic angle spinning carbon-13 NMR of tobacco mosaic virus. An application of the high-resolution solid-state NMR spectroscopy to very large biological systems.

Magic angle spinning 13C NMR was used to study tobacco mosaic virus (TMV) in solution. Well-resolved 13C NMR spectra were obtained, in which several carbon resonances of amino acids of the TMV coat protein subunits that are not observable by conventional high-resolution NMR spectroscopy can be designed. RNA resonance were absent, however, in the magic angle spinning 13C NMR spectra. Since three different binding sites are available for each nucleotide of the RNA, this is probably due to a line broadening caused by distributions of isotropic chemical shift values. In 13C-enriched TM 13C-13C dipolar interactions also gave rise to line broadening. By suitable pulse techniques that discriminate carbon resonances on the basis of their T1 and T1 rho values, it was possible to select particular groups of carbon nuclei with characteristic motional properties. Magic angle spinning 13C NMR spectra obtained with these pulse techniques are extremely well resolved.     

In-situ identification of major metabolites in the red alga Gracilariopsis lemaneiformis using high-resolution magic angle spinning nuclear magnetic resonance spectroscopy

The content of low-molecular-weight compounds in the red alga Gracilariopsis lemaneiformis [(Bory) Dawson, Acleto, et Foldvik] has been analysed in-situ using high-resolution magic angle spinning (HR-MAS) nuclear magnetic resonance (NMR) spectroscopy. The major heteroside was shown to be floridoside, but digeneaside and isofloridoside were also detected in the alga. Other major components were isethionic acid and the amino acids taurine and citrulline. The results from the HR-MAS NMR analysis were confirmed with high-resolution NMR spectroscopy, high-resolution fast atom bombardment mass spectrometry (FABMS) and GC-MS, on material isolated from the studied alga, but also on authentic samples. Received: 2 February 1998 / Accepted: 9 April 1998     

Microfabricated Inserts for Magic Angle Coil Spinning (MACS) Wireless NMR Spectroscopy

This article describes the development and testing of the first automatically microfabricated probes to be used in conjunction with the magic angle coil spinning (MACS) NMR technique. NMR spectroscopy is a versatile technique for a large range of applications, but its intrinsically low sensitivity poses significant difficulties in analyzing mass- and volume-limited samples. The combination of microfabrication technology and MACS addresses several well-known NMR issues in a concerted manner for the first time: (i) reproducible wafer-scale fabrication of the first-in-kind on-chip LC microresonator for inductive coupling of the NMR signal and reliable exploitation of MACS capabilities; (ii) improving the sensitivity and the spectral resolution by simultaneous spinning the detection microcoil together with the sample at the "magic angle" of 54.74° with respect to the direction of the magnetic field (magic angle spinning - MAS), accompanied by the wireless signal transmission between the microcoil and the primary circuit of the NMR spectrometer; (iii) given the high spinning rates (tens of kHz) involved in the MAS methodology, the microfabricated inserts exhibit a clear kinematic advantage over their previously demonstrated counterparts due to the inherent capability to produce small radius cylindrical geometries, thus tremendously reducing the mechanical stress and tearing forces on the sample. In order to demonstrate the versatility of the microfabrication technology, we have designed MACS probes for various Larmor frequencies (194, 500 and 700 MHz) testing several samples such as water, Drosophila pupae, adamantane solid and LiCl at different magic angle spinning speeds.     

Identifying anisotropic constraints in multiply labeled bacteriorhodopsin by 15N MAOSS NMR: a general approach to structural studies of membrane proteins

Structural models of membrane proteins can be refined with sets of multiple orientation constraints derived from structural NMR studies of specifically labeled amino acids. The magic angle oriented sample spinning (MAOSS) NMR approach was used to determine a set of orientational constraints in bacteriorhodopsin (bR) in the purple membrane (PM). This method combines the benefits of magic angle spinning (MAS), i.e., improved sensitivity and resolution, with the ability to measure the orientation of anisotropic interactions, which provide important structural information. The nine methionine residues in bacteriorhodopsin were isotopically (15)N labeled and spectra simplified by deuterium exchange before cross-polarization magic angle spinning (CPMAS) experiments. The orientation of the principal axes of the (15)N chemical shift anisotropy (CSA) tensors was determined with respect to the membrane normal for five of six residual resonances by analysis of relative spinning sideband intensities. The applicability of this approach to large proteins embedded in a membrane environment is discussed in light of these results.     

Adiabatic Heteronuclear Decoupling in Rotating Solids

In magic angle spinning solid state NMR experiments the potential of heteronuclear (1)H decoupling employing a continuous train of adiabatic inversion pulses has been assessed via numerical simulations and experimental measurements. It is shown that, with a (1)H RF field strength of approximately 100 kHz that is typically available in MAS NMR probes, it is possible to achieve efficient adiabatic (1)H decoupling at low magic angle spinning frequencies. It is pointed out that in the presence of H (1) inhomogeneities it will be advantageous to employ adiabatic decoupling in MAS solid state NMR experiments.     

Measurement of Scaled Residual Dipolar Couplings in Proteins Using Variable-angle Sample Spinning

NMR spectra of ubiquitin in the presence of bicelles at a concentration of 25% w/v have been recorded under sample spinning conditions for different angles of rotation. For an axis of rotation equal to the magic angle, the (1)H/(15)N HSQC recorded without any (1)H decoupling in the indirect dimension corresponds to the classical spectrum obtained on a protein in an isotropic solution and allows the measurement of scalar J-couplings (1) J (NH). For an angle of rotation smaller than the magic angle, the bicelles orient with their normal perpendicular to the spinning axis, whereas for an angle of rotation greater than the magic angle the bicelles orient with their normal along the spinning axis. This bicelle alignment creates anisotropic conditions that give rise to the observation of residual dipolar couplings in ubiquitin. The magnitude of these dipolar couplings depends directly on the angle that the rotor makes with the main magnetic field. By changing this angle in a controlled manner, residual dipolar couplings can be either scaled up or down thus offering the possibility to study simultaneously a wide range of dipolar couplings in the same sample.     

High-resolution 3D structure determination of kaliotoxin by solid-state NMR spectroscopy

High-resolution solid-state NMR spectroscopy can provide structural information of proteins that cannot be studied by X-ray crystallography or solution NMR spectroscopy. Here we demonstrate that it is possible to determine a protein structure by solid-state NMR to a resolution comparable to that by solution NMR. Using an iterative assignment and structure calculation protocol, a large number of distance restraints was extracted from (1)H/(1)H mixing experiments recorded on a single uniformly labeled sample under magic angle spinning conditions. The calculated structure has a coordinate precision of 0.6 A and 1.3 A for the backbone and side chain heavy atoms, respectively, and deviates from the structure observed in solution. The approach is expected to be applicable to larger systems enabling the determination of high-resolution structures of amyloid or membrane proteins.     

High-resolution magic angle spinning nuclear magnetic resonance analysis of metabolic changes in melanoma cells after induction of melanogenesis

Melanin synthesis affects melanoma behavior and tumor responsiveness to therapy; therefore, we investigated metabolic changes in melanoma cells after induction of melanogenesis. Amelanotic and melanotic melanoma cells were labeled with ¹³C precursors and changes in their metabolism was analyzed by high-resolution magic angle spinning (HRMAS) nuclear magnetic resonance (NMR). HRMAS NMR demonstrated clear differences in the pattern of metabolic intermediates between amelanotic and melanotic cells. Although the exact nature of the metabolites requires further investigations, our comparative studies clearly show that induction of melanogenesis is associated with changes of glucose and sodium acetate metabolism, demonstrating HRMAS NMR as a powerful and noninvasive technique to investigate such process.     

Structural and dynamic studies of proteins by solid-state NMR spectroscopy: rapid movement forward

Starting only a few years ago, many solid-state NMR spectroscopy laboratories have become engaged in solving the complete structures of biological macromolecules using high-resolution methods based on magic angle spinning. These efforts typically involve structurally homogeneous samples, and utilize recently developed pulse sequences for the sequential correlation of resonances, the detection of tertiary contacts and the characterization of torsion angles. Thereby, systems have been studied that evaded other, more established, structure determination methods.     

A study of a Calpha,beta-didehydroalanine homo-oligopeptide series in the solid-state by 13C cross-polarization magic angle spinning NMR.

The fully extended peptide conformation (2.0(5)-helix) has been investigated for the first time in the solid-state by 13C cross-polarization magic angle spinning NMR. The compounds examined are members of a terminally protected, homo-oligopeptide series (from monomer through hexamer) based on Calpha,beta-didehydroalanine.     

Distinguishing individual lipid headgroup mobility and phase transitions in raft-forming lipid mixtures with 31P MAS NMR

A model membrane system composed of egg sphingomyelin (SM), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and cholesterol was studied with static and magic angle spinning (31)P NMR spectroscopy. This model membrane system is of significant biological relevance since it is known to form lipid rafts. (31)P NMR under magic angle spinning conditions resolves the SM and DOPC headgroup resonances allowing for extraction of the (31)P NMR parameters for the individual lipid components. The isotropic chemical shift, chemical shift anisotropy, and asymmetry parameter can be extracted from the spinning side band manifold of the individual components that form liquid-ordered and liquid-disordered domains. The magnitude of the (31)P chemical shift anisotropy and the line width is used to determine headgroup mobility and monitor the gel-to-gel and gel-to-liquid crystalline phase transitions of SM as a function of temperature in these mixtures. Spin-spin relaxation measurements are in agreement with the line width results, reflecting mobility differences and some heterogeneities. It will be shown that the presence of DOPC and/or cholesterol greatly impacts the headgroup mobility of SM both above and below the liquid crystalline phase transition temperature, whereas DOPC displays only minor variations in these lipid mixtures.     

In situ 19F NMR studies of an E. coli membrane protein

In this report, (19)F spin incorporation in a specific site of a specific membrane protein in E. coli was accomplished via trifluoromethyl-phenylalanine ((19) F-tfmF). Site-specific (19)F chemical shifts and longitudinal relaxation times of diacylglycerol kinase (DAGK), an E. coli membrane protein, were measured in its native membrane using in situ magic angle spinning (MAS) solid state nuclear magnetic resonance (NMR). Comparing with solution NMR data of the purified DAGK in detergent micelles, the in situ MAS-NMR data illustrated that (19)F chemical shift values of residues at different membrane protein locations were influenced by interactions between membrane proteins and their surrounding lipid or lipid mimic environments, while (19)F side chain longitudinal relaxation values were probably affected by different interactions of DAGK with planar lipid bilayer versus globular detergent micelles.     

Static and magic angle spinning (31)P NMR spectroscopy of two natural plasma membranes

Static and magic angle spinning (31)P NMR spectroscopy was used for the first time in natural plasma membranes from erythrocytes and skeletal muscle to study phospholipid arrangement and composition. Typical static powder-like spectra were obtained showing that phospholipids were in a bilayer arrangement. Magic angle spinning narrowed spectra into two components. The first one corresponded to phosphatidylcholine and the second one to the other phospholipids with intensities in agreement with the known phospholipid composition. These findings show that NMR data previously acquired using model membranes can be transposed to studies on phospholipids in their natural environment.     

Structure of the transmembrane dimer interface of glycophorin A in membrane bilayers

The hydrophobic transmembrane domain of glycophorin A contains a sequence motif that mediates dimerization in membrane environments. Long-range interhelical distance measurements using magic angle spinning NMR spectroscopy provide high-resolution structural constraints on the packing of the dimer interface in membrane bilayers. We show that direct packing contacts occur between glycine residues at positions 79 and 83 in the transmembrane sequence. Additional interhelical constraints between Ile76 and Gly79 and between Val80 and Gly83 restrict the rotational orientation and crossing angle of the interacting helices. These results refine our previously proposed structure of the glycophorin A dimer [Smith, S. O., and Bormann, B. J. (1995) Proc. Natl. Acad. Sci. U.S.A. 92, 488-491] which revealed that the methyl groups of Val80 and Val84 are packed against Gly79 and Gly83, respectively.     

Structural studies of the O-specific polysaccharide from Plesiomonas shigelloides strain CNCTC 113/92.

The structure of the O-specific side chain of the lipopolysaccharide (LPS) of Plesiomonas shigelloides, strain CNCTC 113/92 has been investigated by NMR spectroscopy, matrix-assisted laser desorption/ionization time of flight mass spectrometry and sugar and methylation analysis. It was concluded that the polysaccharide is composed of a hexasaccharide repeating unit with the following structure: in which D-beta-D-Hepp is Dglycero-beta-Dmanno-heptopyranose and 6d-beta-D-Hep is 6-deoxy-beta-Dmanno-heptopyranose. This structure represents a novel hexasaccharide repeating unit of bacterial O-antigen that is characteristic and unique to the Plesiomonas shigelloides strain. Using the high-resolution magic angle spinning technique, 1H-NMR spectra were also obtained for the O-polysaccharide components of isolated LPS and in their original form directly on the surface of bacterial cells.     

Applications of Variable-angle Sample Spinning Experiments to the Measurement of Scaled Residual Dipolar Couplings and <Superscript>15</Superscript>N CSA in Soluble Proteins

NMR spectra of ubiquitin in the presence of bicelles at a concentration of 32% w/v have been recorded at 700 MHz under sample spinning conditions at the magic angle (54.7°) and at an angle of 45.5°. At the magic angle, the ¹H–¹⁵N HSQC spectrum of ubiquitin in bicelles is virtually indistinguishable from the one recorded on the protein in solution. Spinning the sample at the magic angle creates an isotropic environment with no preferred bicelle orientations, thus allowing the determination of scalar coupling constants. For an angle of rotation of 45.5°, the bicelles orient with their normal perpendicular to the spinning axis leading to the observation of strong residual dipolar couplings and chemical shift variations of the ¹⁵N resonances. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.