Joel R. Garbow

Quantitative determination of the concentrations of 13C15N chemical bonds by double cross-polarization NMR

Methode de determination des liaisons chimiques entre 15 N et 13 C dans des composes doublement marques par ces isotopes en utilisant la double polarisation croisee en RMN

Analysis of chitin structure by nuclear magnetic resonance spectroscopy and chitinolytic enzyme digestion.

Solid-state 13C-NMR analysis of chitin prepared from cuticle of the tobacco hornworm, Manduca sexta (L.), and of crab yielded spectra that demonstrate a high degree of chemical homogeneity (greater than 95%) for the preparations. The chemical shifts of the well-resolved carbon signals from both samples matched closely those of the monomeric unit 2-acetamido-2-deoxy-D-glucopyranoside (GlcNAc). Chromatographic analysis of products from the digestion of chitin by the binary chitinase system (endo splitting chitinase and exo splitting beta-N-acetylglucosaminidase) isolated from M. sexta molting fluid showed that the major product from both chitin preparations is GlcNAc. Also detected was a minor product (product U) that had a chromatographic retention time on the carbohydrate analysis column intermediate between those of chitin penta- and hexasaccharides. Gel filtration chromatography of U indicated that U had an apparent molecular weight intermediate between that of GlcNAc and of N,N-diacetylchitobiose. Cation-exchange chromatography of U after acid hydrolysis revealed the presence of glucosamine only. Derivatization with trinitrobenzenesulfonate showed the presence of a free amino group in U. Solution proton and carbon NMR spectroscopy were used to identify U as a N-monoacetylchitobiose [O-beta-D-2-amino-2-deoxyglucopyranosyl- (1----4)-2-acetamido-2-deoxy-beta-D-glucopyranose] with the residue at the nonreducing end deacetylated. These studies showed that chitin prepared from alkali- and heat-treated insect or crab cuticle contains trace levels of deacetylated residues that are released as a dead-end product, N-monoacetylchitobiose, after digestion by the binary enzyme system.

NMR studies of structure and dynamics in fruit cuticle polyesters

Cutin and suberin are support polymers involved in waterproofing the leaves and fruits of higher plants, regulating the flow of nutrients among various plant organs, and minimizing the deleterious impact of microbial pathogens. Despite the complexity and intractable nature of these plant biopolyesters, their molecular structure and development are amenable to study by suitable solid-state and solution-state NMR techniques. Interactions of tomato cutin with water were examined by solid-state 2H and 13C NMR, showing that water films enhance rapid segmental motions of the acyl chains and are associated with a fivefold increase in surface elasticity upon cutin hydration. The suberization of wounded potato tissues was studied by solid-state 13C NMR, revealing the likely phenylpropanoid structures that permit dense cross-linking of the suberin structure and their proximity to the cell-wall polysaccharides. Finally, two new approaches were developed to elucidate the molecular structures of these biopolymers: partial depolymerization followed by spectroscopic analysis of the soluble oligomers; and swelling of the intact materials followed by magic-angle spinning (MAS) NMR analysis.

High-resolution NMR in inhomogeneous fields

Abstract A new NMR technique is presented that yields high-resolution, 1-D NMR spectra of solutes in inhomogeneous magnetic fields. The method exploits the nuclear Overhauser effect which couples the longitudinal relaxation of solvent and solute nuclear spins. Effectively, the solvent spins serve as reporters or local gaussmeters for the solute spins. Both 2-D and 1-D versions of this new experiment are reported. Potential applications of the method include in vivo NMR spectroscopy, where the field homogeneity is alwats degraded by the magnetic susceptibilities of the various tissues.

Nondestructive NMR determination of oil composition in transformed canola seeds.

Magic-angle spinning (MAS) 13C nuclear magnetic resonance (NMR) spectroscopy is a convenient method for nondestructive, quantitative characterization of seed oil composition. We describe results for intact hybrid and transformed canola seeds. The MAS 13C NMR technique complements and agrees with gas chromatography results. The spectral resolution approaches that of neat, liquid oils. MAS 13C NMR data allow quantitative analysis of major oil components, including saturates and oleic, linoleic, and linolenic acyl chains. 13C NMR directly and quantitatively elucidates, triglyceride regiochemistry and acyl chain cis-trans isomers that cannot be quickly detected by other methods. MAS 13C NMR can serve as the primary method for development of near-infrared seed oil calibrations. These NMR methods are nondestructive and attractive for plant-breeding programs or other studies (e.g., functional genomics) where loss of seed viability is inconvenient.

The importance of precise timing in pulsed, rotor-synchronous MAS NMR

Abstract Magic-angle spinning NMR experiments with synchronously applied pulse trains are used to measure a variety of different anisotropic interactions, such as heteronuclear dipolar couplings and chemical-shift anisotropies. When the sample rotation speed and the pulse-sequence timings are not properly synchronized, the resulting spectra may contain artifacts and anisotropic interactions may be measured incorrectly. Sample-rotation/pulse-sequence asynchrony can result from short-term fluctuations in spinning speed, even when the average spinning speed is carefully regulated. Here we examine three simple, spin-echo pulse sequences under conditions where the pulse trains are incommensurate with the sample rotation.

Long-distance rotational echo double resonance measurements for the determination of secondary structure and conformational heterogeneity in peptides

The utility of rotational echo double resonance (REDOR) NMR spectroscopy for determining the conformations of linear peptides has been examined critically using a series of crystalline and amorphous samples. The focus of the present work was the evaluation of long-distance (> 5 A) interactions using 13C-15N dephasing. Detailed studies of specifically labeled melanostatin and synthetic analogs of the alpha-factor yeast mating hormone show that nitrogen-dephased, carbon-observe REDOR measurements are reliable for distances up to 6.0 A, and that dipolar interactions can be detected for distances up to 7 A. By contrast, nitrogen-observe REDOR gives reliable results only for distances shorter than 5.0 A. To measure distances accurately, REDOR data must be corrected for the effects of natural-abundance spins. These corrections are particularly important for measuring long distances, which are of the greatest value for determining peptide secondary structure. We have developed a spherical shell model for calculating the effect of these background spins. The REDOR studies also indicate that in a lyophilized powder, the tridecapeptide alpha-factor mating pheromone from Saccharomyces cerevisiae (WHWLQLKPGQPMY) probably exists as a distribution of different turn structures around the KPGQ region. This finding revises previous solid-state NMR studies on this peptide, which concluded alpha-factor assumes a distorted type-I beta-turn in the Pro-Gly central region of the molecule [J.R. Garbow, M. Breslav, O. Antohi, F. Naider, Biochemistry, 33 (1994) 10094].

Characterization of peptidoglycan stem lengths by solid-state 13C and 15N NMR

Lyophilized whole cells of Aerococcus viridans (Gaffkya homari) grown on a synthetic medium containing D-[2-13C, 15N]Ala, or containing both L-[1-13C]Lys and D-[15N]Ala, have been examined by double cross-polarization magic-angle spinning 13C and 15N nuclear magnetic resonance. Results from the double-labeled alanine experiment confirm the absence of metabolic scrambling of alanine by A. viridans. Results from the combined single-label experiment can be used to count directly the number of adjacent L-Lys and D-Ala units in peptide chains of cell-wall peptidoglycan. This count leads to the conclusion that there are no terminal D-Ala or D-Ala-D-Ala units in uncross-linked chains of the peptidoglycan of A. viridans.

Magic-angle 13C NMR analysis of hard wheat flour and dough.

Samples of hard wheat flour and dough are analyzed by magic-angle spinning 13C NMR spectroscopy. Cross-polarization magic-angle spinning (CPMAS) 13C NMR spectra of the dry flour allow its starch and protein content to be accurately measured. These two components are phase-separated. Spectra of hydrated hard wheat doughs are collected under both CPMAS and single-pulse carbon with low-power 1H decoupling conditions. The former report on the macromolecular components of the dough, while the latter reveal small molecules which are solubilized by the water. Results of the present study are interpreted as indicating that the protein is largely unaffected by the added water and remains phased-separated from the starch, while water causes significant changes in polymer dynamics of the starch component.

Long-distance REDOR NMR measurements for determination of secondary structure and conformational heterogeneity in peptides

High-resolution NMR spectroscopy of liquids has become a standard method for conformational studies in biochemistry. This method provides information on the secondary and tertiary structure of macromolecules and on intermolecular interactions such as ligand receptor binding. These determinations are based mainly on interatomic distance measurements by quantitative analysis of NOESY spectra and, to a lesser degree, on torsion angle measurements based on homoor heteronuclear scalar coupling constants. As with liquids, internuclear distances in solids are reflected in dipole-dipole coupling constants between the nuclear magnetic dipoles. Rotational-echo double resonance (REDOR) spectroscopy has been employed to determine heteronuclear dipole-dipole coupling constants [1,2] and to study internuclear distances in peptides, proteins and bound ligands. An important target for the REDOR experiment is an amorphous solid. For biologically important amorphous solids that cannot be obtained in crystalline form, NMR spectroscopy is the only method for direct investigation of conformation at the atomic level. In the present study we examine the REDOR of selectively labeled peptide hormone in lyophilized powders. In order to understand the contribution of naturalabundance nuclei to the REDOR data at long evolution times, we have used a three-body calculation [3,4] and assumed a spherical distance distribution for natural abundance corrections. In addition we have evaluated the REDOR results in terms of a superposition of different distances with appropriate weighting factors. These approaches allow us to fit the experimental data on the to a high degree of accuracy and to conclude that this peptide has a tendency to be bent in a lyophilized powder.