Douglas C. McCain

The solution conformation of sucrose: concentration and temperature dependence

13C-N.m.r. spin-lattice relaxation measurements were used to study molecular motion in aqueous sucrose. Results show that sucrose tumbles anisotropically in solution, and that its conformation is independent of temperature and concentration. Internal rotation occurs with distinctly different rates and activation energies in the three hydroxymethyl groups. Our data are consistent with results of calculations by Bock and Lemieux who predicted that the conformation of aqueous sucrose is similar to the crystal conformation but with the loss of one intramolecular hydrogen-bond.

Internal motions of the three hydroxymethyl groups in aqueous sucrose

Abstract 13C NMR spin-lattice relaxation times and nuclear Overhauser enhancements were measured as a function of temperature, concentration, and magnetic field strength for sucrose solutions in D2O. Using these data, we determined the spectral density functions for 1H13C dipole-dipole interactions at each of the three hydroxymethyl groups in sucrose. The experimental results showed that the frequency dependence of the spectral densities is characterized by the same correlation time as that for overall molecular rotation, but rapid internal motions reduce the spectral density amplitudes. Model calculations suggest that the internal motions may be described as torsional librations.

Water permeability of chloroplast envelope membranes: In vivo measurement by saturation-transfer NMR

In tulip tree (Liriodendron tulipifera) leaves, the proton NMR signal from chloroplast water is resolved from that of water in other leaf compartments. We used the saturation‐transfer NMR method to measure the mean water molecule residence time within a chloroplast, (88 ± 17) ms at 20°C. From the measured chloroplast dimensions, we calculate an effective permeability coefficient of (9 ± 2) × 10−4 for the chloroplast envelope membrane. This is the first in vivo measurement of chloroplast water permeability.

Relationship of Leaf Spectral Reflectance to Chloroplast Water: Content Determined Using NMR Microscopy

Nuclear magnetic resonance (NMR) microscopy and reflectance spectroscopy were used to evaluate the response of leaf spectral reflectance to chloroplast relative water content (RWC). For sun and shade leaves of maple (Acer platanoides L. var Emerald Queen), NMR determined the distribution of water through the leaf thickness and changes in distribution as leaves dried from 100% RWC to approximately 70%, 50%, and 30% RWC. Chloroplast water could be resolved only in shade leaves. Leaf and chloroplast RWC were similar at leaf RWC of 80% or greater, but chloroplast RWC was as much as 12% greater than leaf RWC when leaf RWC decreased below 50%. In contrast, the RWC of nonchloroplast water compartments was as much as 12% less than leaf RWC. Reflectance responses to leaf water content were greatest in the water absorption bands near 1450 nm, 1950 nm, and 2500 nm wavelengths, and were minimal in the near-infrared from approximately 760–1300 nm. In the 400–760 nm (visible) range, reflectance sensitivity increased substantially as chloroplast RWC decreased. Peak visible reflectance sensitivity occurred near 695 nm and 670 nm for adaxial and abaxial surfaces, respectively, and was more closely related to chloroplast than to nonchloroplast or whole leaf RWC.

Effects of chloride on paramagnetic coupling of manganese in calcium chloride-washed photosystem II preparations

The effect of chloride on paramagnetic coupling of manganese in the oxygen‐evolving complex of CaCl2‐‐washed PS II preparations was examined using Q‐band ESR. When these PS II preparations were depleted of chloride, a strong 6‐line ESR signal characteristic of protein‐bound, uncoupled manganese was observed. Incubation at high chloride concentrations caused the disappearance of this signal. By repeated removal and addition of chloride, the signal could be cycled on and off without loss of bound manganese. When in a chloride‐depleted state, the ESR‐detectable protein‐bound manganese could be removed by treatment with EDTA. Subsequent heating of EDTA‐treated preparations revealed a second pool of protein‐bound manganese associated with PS II. One of these pools requires a high concentration of chloride to maintain paramagnetic coupling while the second pool (within the limits of our observations) does not appear to require chloride for the maintenance of the paramagnetically coupled state.

NMR relaxation study of internal motions in Staphylococcal nuclease

Abstract 13C nuclear spin relaxation times (T1 and T2) were measured for three glycine α-carbon atoms in aqueous staphylococcal nuclease. Three forms of the protein were studied: the uncomplexed nuclease, its calcium complex, and a ternary complex with calcium and an inhibitor. The NMR data were used to calculate order parameters and effective correlation times that describe internal motion. Major sources of experimental error were identified, and statistical analyses were used to examine the accuracy of the method. Results showed that when the inhibitor binds to the protein, a significant increase in stiffness occurs at glycines near the calcium binding site.

Combined Effects of Light and Water-Stress on Chloroplast Volume Regulation

A nuclear magnetic resonance technique was used to measure changes in the water content of Acer platanoides chloroplasts in leaf discs that had reached osmotic equilibrium with external solutions either in the dark or under exposure to light. Results showed that chloroplast volume regulation (CVR) maintained constant water content in the chloroplasts over a range of water potentials in the dark, but CVR failed when the water potential fell below a critical value. The critical potential was lower in the dark in sun leaves than in shade leaves. Upon exposure to intense light, CVR remained effective in sun leaves over the same range as in the dark, but it failed in shade leaves at all water potentials. Osmolytes are necessary for CVR, but KCl is relatively ineffective; increased concentrations of intracellular KCl did not fully support an increase in the range of CVR. The results indicate that leaves need reserve supplies of cytosolic osmolytes to maintain CVR at low water potentials, and a larger reserve supply is needed in leaves that are exposed to intense light.

A theory and a model for interpreting the proton nuclear magnetic resonance spectra of water in plant leaves

Some plant leaves display complex, orientation-dependent, proton nuclear magnetic resonance (1H NMR) spectra. The spectral patterns vary as the angle between the leaf surface and the applied magnetic field is varied. They also vary with temperature and with the quantity of absorbed manganous ions, but they are independent of magnetic field strength. In this paper, we propose a theory to explain the origin of the spectra and a model from which the patterns can be calculated. The theory shows how heterogeneous magnetic susceptibilities and local dipolar magnetic fields in chloroplasts can shift the water-proton resonance field. The model describes a simplified leaf structure in which the chloroplasts are nonrandomly aligned with respect to the leaf surface. Model calculations are tested by comparison with experimental spectra from hawthorn leaves (Crataegus sp.).

In vivo study of chloroplast volume regulation

This paper describes a new technique that can be used to study chloroplast volume regulation in vivo. Nuclear magnetic resonance spectroscopy was used to measure relative amounts of chloroplast water in Acer platanoides leaves as they dried in air, and also in leaf disks exposed to aqueous polyethylene glycol, sucrose, or glycerol. The chloroplasts retained a constant quantity of water as leaf water potentials varied between -0.05 and -1.90 MPa, indicating that volume regulation was effective throughout this range. The chloroplasts lost water when the water potential fell below -1.90 MPa, except when leaf disks were exposed to glycerol, suggesting that the lower limit of effective volume regulation is determined by physiological levels of osmotic solutes and that glycerol can be used for chloroplast osmoregulation.

Carbon-13 NMR study of southern bean mosaic virus.

The motional state of the coat protein of southern bean mosaic virus (SBMV) particles has been studied by 13C NMR at 50.3 MHz. In the native virus, 13C-NMR signals are observed only from a few highly mobile amino acid side chain groups such as methyls or from unprotonated carbons such as carbonyls or arginine-zeta. After swelling SBMV by treating with EDTA, additional 13C-NMR peaks become visible. The pattern of resonances matches that expected from the side chains of those amino acid residues identified to be on the surface of the virus particle based on the known crystal structure. After further disruption of the virus structure by 0.5 M KCl, the 13C-NMR peaks sharpen, and additional peaks appear, but the set of peaks appears to correspond only to surface amino acid side chains.