An NMR method to characterize multiple water compartments on mammalian collagen

A molecular model is proposed to explain water 1H NMR spin-lattice relaxation at different levels of hydration (NMR titration method) on collagen. A fast proton exchange model is used to identify and characterize protein hydration compartments at three distinct Gibbs free energy levels. The NMR titration method reveals a spectrum of water motions with three well-separated peaks in addition to bulk water that can be uniquely characterized by sequential dehydration. Categorical changes in water motion occur at critical hydration levels h (g water/g collagen) defined by integral multiples N = 1, 4 and 24 times the fundamental hydration value of one water bridge per every three amino acid residues as originally proposed by Ramachandran in 1968. Changes occur at (1) the Ramachandran single water bridge between a positive amide and negative carbonyl group at h1 = 0.0658 g/g, (2) the Berendsen single water chain per cleft at h2 = 0.264 g/g, and (3) full monolayer coverage with six water chains per cleft level at h3 = 1.584 g/g. The NMR titration method is verified by comparison of measured NMR relaxation compartments with molecular hydration compartments predicted from models of collagen structure. NMR titration studies of globular proteins using the hydration model may provide unique insight into the critical contributions of hydration to protein folding.     

核磁共振技术在土壤-植物-大气连续体研究中的应用

植物体内的水分状态与传输过程是土壤-植物-大气连续体(SPAC)水分传输理论的核心内容,也是研究植物水分利用与调控的基础.植物体内水分的传输过程受外界环境影响较大,植物需要通过对体内水分状态的适当调整来适应环境变化和维持自身的生长发育.由于蒸发通量、压力室、高压流速仪、热脉冲等传统检测方法往往会对植株造成破坏和损伤,因此难以准确反映和定量描述植物体内水分传输的真实过程.核磁共振技术(NMR)由于其无损、非侵入的特点,在植物水分分布和传输相关研究中日益得到关注.本文概述了NMR在检测植物体内水分分布、传输以及含量测定等方面的研究进展,还分析了目前NMR技术在SPAC系统研究中存在的问题及可能的解决方法,并指出NMR技术将来可能在植物水分生理、植物与环境互作以及水分代谢等相关研究领域的应用.NMR技术在SPAC系统研究中的应用在我国仍处于初级阶段,开发户外便携式、开放式检测仪器是NMR技术在SPAC研究领域进一步应用和推广的关键所在.     

Quantitative determination of water in the skin by 1H NMR

Studies were made on determination of the amount of water in rat skin by 1H NMR spectroscopy. The NMR spectrum obtained depended on how the skin was packed into the NMR tube. Consistent results were obtained by packing the skin into a specially designed NMR cell. By this technique the amount of water in the skin could be measured accurately, and results were comparable with those obtained by differential scanning calorimetry. The water content of the skin of rats was consistently about 20% more in females than in males.     

Oxygen-17 and deuterium nuclear magnetic resonance studies of lysozyme hydration

Oxygen-17 and deuterium NMR studies of lysozyme hydration are reported for a wide range of lysozyme concentrations, and the relationship between water "activity" and water mobility in the lysozyme-water system as determined by high-field NMR is examined. In a first approximation, the effect of lysozyme activity on hydration is considered to be small because of the relatively low charge on lysozyme at pH 7 and the absence of salt in the lysozyme solutions. Correlation times are determined for tightly bound water, weakly bound water, and "multilayer" or trapped water in lysozyme at 20 degrees C. Hydration numbers are also determined for these three different water populations interacting with lysozyme. Good agreement is found between the hydration numbers determined by 17O NMR and the calculations based on the D'Arcy and Watt analysis of water sorption isotherms for proteins that considered three major water populations in hydrated lysozyme. A molecular interpretation for the three components in the D'Arcy and Watt theory of sorption isotherms is also proposed on the basis of our NMR results. Previous proton NMR spin-echo results are shown to be consistent with our findings by 17O NMR and support the view that there are at least four regions of distinct hydration behavior of lysozyme which span the whole range from solutions to solid powders.     

A new nuclear magnetic resonance property of lung

Inflated lung has a nuclear magnetic resonance (NMR) free-induction decay (FID) which is short compared with that of collapsed lung and those of other body tissues. An almost identically short FID is obtained from a slurry of 5-micron alumina particles in water. Interfaces between air and water in lung and between alumina and water in the slurry appear to be the source of spatial internal magnetic inhomogeneities which produce NMR line broadening and the short FID. Paired images that included lung, taken with paired symmetric and asymmetric NMR spin-echo sequences, permit the generation of an image, by subtraction, of the lung isolated from surrounding tissue. These new lung images are neither proton density, T1 (spin-lattice relaxation time), nor T2 (spin-spin relaxation time) images. They complement current NMR images and provide information about regional lung inflation. This previously unrecognized NMR property of lung tissue has potential application in NMR imaging, in quantitative determination of lung water and its distribution, and in the quantitation of regional lung inflation.     

Water Balance in Cucumis Plants, Measured by Nuclear Magnetic Resonance, II

Nuclear magnetic resonance (NMR) was used to investigate theeffects of changes in root temperature, of changes in the areaof root in contact with culture solution and of day/night rhythmon the water balance of a cucumber and a gherkin plant. Resultsare discussed in terms of water potential, flow rate and resistanceusing a previously presented model of water balance. As longas water uptake alone is varied, flow rate and water content(or potential) will change in the same direction. In contrast,from that model it is predicted that changes in transpirationwill affect flow rate and water content in opposite ways. Anexperimental verification of this prediction was given in theprevious paper. Results obtained by the NMR method are comparedto those determined using a dendrometer. The results demonstratethat the NMR method is a valuable tool to study plant waterbalance and that it can serve as a technique for discriminatingbetween changes in plant water balance that are due to changesin water uptake by roots and those due to changes in transpiration. Key words: Water balance model, Cucumis satious L., flow, water content, NMR, water balance measurement     

Search for an NMR signal from spin isomers of water in H2O/D2O mixture

With the aim to detect the existence of spin-isomeric molecules in water by NMR tomography, comparative measurement of NMR signal integral amplitude was performed with pure water and 50/50 water/heavy water mixture. Magnetic field was 0.5 T in tomographic and spectroscopic regimes. No spin-isomeric effect was registered, taking into account that the error of measurements did not exceed 5%.     

The determination of intracellular water space by NMR

A new method for the determination of intracellular water space using NMR spectroscopy is described. The method is based on the measurement of 59Co NMR signal intensity of an inert, stable and membrane-impermeable cobalt(III) compound such as Co(CN)3-6 or Co(imidazole)3+6 and the 2H or 1H NMR signal intensities of the freely permeable water. As an example of the method, the variation of the intracellular water space of human erythrocytes as a function of osmolality was measured.     

Movement of water in conjunction with plant movement visualized by NMR imaging

The water distribution in the pulvinus of Mimosa can be visualized by an NMR imaging technique. After stimulation of a Mimosa plant, water in the lower half of the main pulvinus disappeared, the water previously contained in this area seeming to be transferred to the upper half of the main pulvinus. Movement of the water in conjunction with Mimosa movement was visualized sequentially by a non-invasive NMR imaging procedure.     

NMR study of seed hydration with deuterated water: dependence of proton signals on hydration level.

Proton NMR signals in seeds are shown to depend on hydration level. In fact at low water amount, as it occurs in many native seeds, protons can have a restricted mobility and are not detectable. A NMR method for measuring the dependence of proton signals on hydration is reported. The method also allows the separation of the contributions of water and non-water protons in a low-resolution NMR experiment. It is based on successive hydrations (with deuterated water) - desiccation steps and on the analysis of the transverse magnetization decay curves.     

The distribution of water in native starch granules—a multinuclear NMR study

The microscopic distribution and dynamic state of water in native potato, maize and pea starch granules are investigated with NMR relaxometry and diffusometry. Besides extra-granular water, three water populations can be identified inside native potato starch granules. These are assigned to water in the amorphous growth rings; water in the semi-crystalline lamellae and “channel water”, which is located in the hexagonal channels within the B-type amylopectin crystals. The first two water populations are orientationally disordered and exchange with each other on a millisecond timescale at 290 K. NMR diffusometry shows that the water in packed granule beds is undergoing translational diffusion in a 2-dimensional space, either in thin layers between granules and/or in amorphous growth rings within the granules. The “channel water” is uniquely characterised by a 1 kHz deuterium doublet splitting and is in slow exchange with water in the other compartments on the NMR timescale. In the smaller maize granules all intra-granular water populations are in fast exchange and there is no evidence for “channel water” in the A-type crystal lattice. The NMR water proton and deuterium data for pea starch are consistent with a composite A and B-type crystal structure.     

A nuclear magnetic resonance imaging technique designed for studies of water in plant leaves.

A new imaging technique is described which uses nuclear magnetic resonance (NMR) to create a water profile of plant leaves. The water profile shows the average distribution of water as a function of depth into a leaf along a line perpendicular to the leaf surface; it can be used to measure the thickness of cell layers and the quantity of water in each layer. Two-dimensional NMR methods were used to avoid chemical shift distortion which degrades the resolution in leaf images made by conventional NMR techniques; image resolution was improved further by deconvolution analysis. To illustrate its application, the technique was used to follow changes in the internal structure of developing leaves.     

In Situ Investigation of Leaf Water Status by Portable Unilateral Nuclear Magnetic Resonance

A portable unilateral nuclear magnetic resonance (NMR) instrument was used to detect in field conditions the water status of leaves of herbaceous crops (Zea mays, Phaseolus vulgaris), mesophyllous trees (Populus nigra), and natural Mediterranean vegetation characterized by water-spending shrubs (Cistus incanus) and water-saving sclerophyllous trees (Quercus ilex). A good relationship was observed between NMR signal, leaf relative water content, and leaf transpiration in herbaceous leaves undergoing fast dehydration or slowly developing a drought stress. A relationship was also observed between NMR signal and water potential of Populus leaves during the development of a water stress and when leaves recovered from the stress. In the natural vegetation, the relationship between NMR signal and water status was found in Cistus, the species characterized by high transpiration rates, when measured during a drought stress period and after a rainfall. In the case of the sclerophyllous Quercus, the NMR signal, the relative water content, and the transpiration rate did not change at different leaf water status, possibly because a large amount of water is compartmentalized in cellular structures and macromolecules. The good association between NMR signal and relative water content was lost in leaves exposed for 24 h to dehydration or to an osmotic stress caused by polyethylene glycol feeding. At this time, the transverse relaxation time became longer than in leaves maintained under optimal water conditions, and two indicators of membrane damage, the ion leakage and the emission of products of membrane lipoxygenation [(Z)-3-hexenal, (Z)-3-hexenol, and (E)-2-hexenol], increased. These results taken all together give information on the physiological state of a leaf under a developing stress and show the usefulness of the NMR instrumentation for screening vegetation health and fitness in natural and cultivated conditions. It is concluded that the portable unilateral NMR instrument may be usefully employed in field conditions to monitor nondestructively the water status of plants and to assist agricultural practices, such as irrigation scheduling, to minimize stomatal closure and the consequent limitation to plant production.     

The interaction of water molecules with purple membrane suspension using 2H double-quantum filter, 1H and 2H diffusion nuclear magnetic resonance

Bacteriorhodopsin is a membrane protein of the purple membrane (PM) of Halobacterium salinarum, which is isolated as sheets of highly organized two-dimensional hexagonal microcrystals and for which water molecules play a crucial role that affects its function as a proton pump. In this paper we used single- and double-quantum (2)H NMR as well as (1)H and (2)H diffusion NMR to characterize the interaction of water molecules with the PM in D(2)O suspensions. We found that, under the influence of a strong magnetic field on a concentrated PM sample (0.61 mM), the PM sheets affect the entire water population and a residual quadrupolar splitting (upsilon(q) approximately 5.5 Hz, 298 K, at 11.7 T) is observed for the D(2)O molecules. We found that the residual quadrupolar coupling, the creation time in which a maximal DQF signal was obtained (tau(max)), and the relative intensity of the (2)H DQF spectrum of the water molecules in the PM samples (referred to herein as NMR order parameters) are very sensitive to temperature, dilution, and chemical modifications of the PM. In concentrated PM samples in D(2)O, these NMR parameters seem to reflect the relative organization of the PM. Interestingly, we have observed that some of these parameters are sensitive to the efficiency of the trimer packing, as concluded from the apo-membrane behavior. The data for dionized blue membrane, partially delipidated sample, and detergent-treated PM show that these D(2)O NMR order parameters, which are magnetic field dependent, are sensitive to the structural integrity of the PM. In addition, we revealed that heating the PM sample inside or outside the NMR magnet has, after cooling, a different effect on the NMR characteristics of the water molecules in the concentrated PM suspensions. The difference in the D(2)O NMR order parameters for the PM samples, which were heated and cooled in the presence and in the absence of a strong magnetic field, corroborates the conclusions that the above D(2)O order parameters are indirect reflections of both microscopic and macroscopic order of the PM samples. In addition, (1)H NMR diffusion measurements showed that at least three distinct water populations could be identified, based on their diffusion coefficients. These water populations seem to correlate with different water populations previously reported for the PM system.     

Determination of the structure of [Nle7]-endothelin by 1H NMR

[Nle7]-endothelin was synthesized and studied by 1H NMR and distance geometry calculations. The NMR study was performed first in DMSO-d6 and then in 50% acetonitrile/water since this peptide aggregates in pure water. In both cases, all spin systems were identified and assigned with the aid of two-dimensional spectroscopy (2D): COSY (for scalar couplings) and NOESY (for dipolar couplings). On the basis of the acetonitrile/water NMR parameters, and using the DISGEO program, a three-dimensional structure of [Nle7]-endothelin is proposed and discussed.     

Use of NMR for measurement of total body water and estimation of body fat

A nuclear magnetic resonance (NMR) method is described for quantitatively measuring total body water (TBW) and for estimating the fat content of baboons. The hydrogen associated with water was measured as the amplitude of the free-induction decay voltage following a series of 90 degree radio frequency pulses at the Lamour frequency for hydrogen with a pulse length of 14 microseconds and a peak measuring time of 50 microseconds. TBW was calculated by multiplying the peak amplitude (volts) by the experimentally determined constant for a water standard (g water/V). This NMR method yielded TBW contents similar to those obtained in the same baboons by direct gravimetric procedures. In contrast, the widely used 3H2O-dilution method usually and variably overestimated body water. By providing an accurate measure of body water, this NMR procedure provides a rapid, noninvasive, reasonably accurate way of estimating body fat content.     

Water exchange in plant tissue studied by proton NMR in the presence of paramagnetic centers.

The proton NMR relaxation of water in maize roots in the presence of paramagnetic centers, Mn2+, Mn- EDTA2 -, and dextran-magnetite was measured. It was shown that the NMR method of Conlon and Outhred (1972, Biochem. Biophys. Acta. 288:354-361) can be applied to a heterogenous multicellular system, and the water exchange time between cortical cells and the extracellular space can be calculated. The water exchange is presumably controlled by the intracellular unstirred layers. The Mn- EDTA2 - complex is a suitable paramagnetic compound for complex tissue, while the application of dextran-magnetite is probably restricted to studies of water exchange in cell suspensions. The water free space of the root and viscosity of the cells cytoplasm was estimated with the use of Mn- EDTA2 -. The convenience of proton NMR for studying the multiphase uptake of paramagnetic ions by plant root as well as their transport to leaves is demonstrated. A simple and rapid NMR technique (spin-echo recovery) for continuous measurement of the uptake process is presented.     

Ribonuclease A--a complete proton NMR fingerprint using TOCSY and NOESY experiments in water

In our attempts to obtain total sequential assignment of the 1H NMR spectrum of ribonuclease A, several published water suppression techniques were tested and assessed. The jump- and-return sequence and its echo hybrid were used with considerable success in both the TOCSY and NOESY experiments on 3mM ribonuclease A solutions. The NMR approach used here may be of general applicability for 1H NMR studies of proteins in water of concentrations under 5mM.     

A 1H NMR study of the solution conformation of the neuropeptide galanin.

The conformations of the neuropeptide galanin in water and trifluoroethanol solutions have been examined by 1H NMR spectroscopy. Analysis of two-dimensional NMR experiments enabled the assignment of virtually all the 1H resonances of galanin in trifluoroethanol solution and many of the 1H resonances in aqueous solution. Interpretation of the NMR data in structural terms suggests that in trifluoroethanol galanin is predominantly helical while in water it does not adopt a fixed conformation.     

水合溶菌酶及其热稳定性的NMR研究

本文用90MHz脉冲NMB波谱仪记录了具不同含水量的溶菌酶的质子宽谱线NMR谱图及自由感应衰减曲线,并记录了水合度为0.10及0.19克水/克溶菌酶的溶菌酶样品从室温到热变性温度范围内的谱图.用线宽参量随水合度及温度的变化讨论溶菌酶在水合及热变性过程中溶菌酶分子及水分子运动性的变化.结果表明,溶菌酶分子及水分子的运动性与水合溶菌酶中的水含量密切相关;低水含量的水合溶菌酶在热变性过程中酶分子运动性的变化经历了两个转变,分别对应于酶分子间的解缔合及分子内的解旋.