Measurements of water proton NMR spin-lattice relaxation time in the rotating frame (T1p) for studying motions in solutions of giant macro-molecules and supramolecular particles (T2 virus)

Spin-spin relaxation time (T2), spin-lattice relaxation time (T1), and spin-lattice relaxation time in the rotating frame (T1p) of water protons in solutions of bacteriophage T2 were studied by pulsed nuclear magnetic resonance. The frequency dependence of the measurements exhibits a dispersion implying existence of a fraction of water molecules in solution with a correlation time distribution centered at approximately 10(-5) sec which is strongly influenced by the reorientational motions of virus particles. Experiments were carried out with two forms of bacteriophage T2 existing at pH 5.4 and 7.8 respectively. The different structures of the virus at these two pH values are reflected in the NMR relaxation behavior of water protons.     

The molecular mobility of water in natural polymers: silk Bombyx mori with a low water content as studied by 1H DQF NMR

The molecular mobility of water in fibres of natural silk (Bombyx mori) was studied by the double-quantum-filtered (DQF) and single-pulse 1H NMR techniques. The results obtained showed a slow motion of water molecules and their strong interaction with silk macromolecules. At different model functions for resonance lineshape in 1H NMR spectra, the influence of signal linewidth on the estimation of relaxation times and cross-relaxation parameters was considered. The observed 1H DQF NMR signal in B. mori silk fibres (BC = 0.065) indicated a local order and anisotropic motion of water molecules, which leads to 1H-1H dipolar interactions in natural silk fibers due to the creation of the second-rank tensors (T(2,+1), T(2,-1)). DQF spectra were the difference of two Lorentzians with different linewidths and were analyzed using the theory of 1H DQF NMR and the data on residual dipolar interactions in systems with the anisotropic mobility of water molecules. The residual dipolar interactions was insignificant and, as the humidity increased (0.18), no DQF-signals and residual dipolar interactions were observed.     

Freezing of phosphocholine headgroup in fully hydrated sphingomyelin bilayers and its effect on the dynamics of nonfreezable water at subzero temperatures.

Differential scanning calorimetry (DSC) and nuclear magnetic resonance (NMR) spectroscopy are applied to characterize the nonfreezable water molecules in fully hydrated D2O/sphingomyelin at temperatures below 0 degrees C. Upon cooling, DSC thermogram displays two thermal transitions peaked at -11 and -34 degrees C. The high-temperature exothermic transition corresponds to the freezing of the bulk D2O, and the low-temperature transition, which has not previously been reported, can be ascribed to the freezing of the phosphocholine headgroup in the lipid bilayer. The dynamics of nonfreezable water are also studied by 2H NMR T1 (spin-lattice relaxation time) and T2e (spin-spin relaxation time obtained by two pulse echo) measurements at 30.7 MHz and at temperatures down to -110 degrees C. The temperature dependence of the T1 relaxation time is characterized by a distinct minimum value of 2.1 +/- 0.1 ms at -30 degrees C. T2e is discontinuous at temperature around -70 degrees C, indicating another freezing-like event for the bound water at this temperature. Analysis of the relaxation data suggest that nonfreezable water undergoes both fast and slow motions at characteristic NMR time scales. The slow motions are affected when the lipid headgroup freezes.     

Low-frequency motion in membranes. The effect of cholesterol and proteins

Nuclear magnetic resonance (NMR) relaxation techniques have been used to study the effect of lipid-protein interactions on the dynamics of membrane lipids. Proton enhanced (PE) 13C-NMR measurements are reported for the methylene chain resonances in red blood cell membranes and their lipid extracts. For comparison similar measurements have been made of phospholipid dispersions containing cholesterol and the polypeptide gramicidin A+. It is found that the spin-lattice relaxation time in the rotating reference frame (T1 rho) is far more sensitive to protein, gramicidin A+ or cholesterol content than is the laboratory frame relaxation time (T1). Based on this data it is concluded that the addition of the second component to a lipid bilayer produces a low-frequency motion in the region of 10(5) to 10(7) Hz within the membrane lipid. The T1 rho for the superimposed resonance peaks derived from all parts of the phospholipid chain are all influenced in the same manner suggesting that the low frequency motion involves collective movements of large segments of the hydrocarbon chain. Because of the molecular co-operativity implied in this type of motion and the greater sensitivity of T1 rho to the effects of lipid-protein interactions generally, it is proposed that these low-frequency perturbations are felt at a greater distance from the protein than those at higher frequencies which dominate T1.     

Use of relaxation-edited one-dimensional and two dimensional nuclear magnetic resonance spectroscopy to improve detection of small metabolites in blood plasma

The 1H nuclear magnetic resonance (NMR) spectra of biological samples, such as blood plasma and tissues, are information rich but data complex owing to superposition of the resonances from a multitude of different chemical entities in multiple-phase compartments, hampering detection and subsequent resonance assignments. To overcome these problems, several spectral-editing NMR experiments are described here, combining spin-relaxation filters (based on T(1), T(rho), and T(2)) with both one-dimensional and two-dimensional (2D) NMR spectroscopy. These techniques enable the separation of NMR resonances based on their relaxation times and allow simplification of the complex spectra. In this paper, the approach is exemplified using a control human blood plasma, which is a complex mixture of proteins, lipoproteins, and small-molecule metabolites. In the case of T(1rho)- and T(2)-edited 2D NMR experiments, a "flip-back" pulse was introduced after the relaxation editing to make the phase cycling of the "relaxation filter" and the 2D NMR part independent, thus enabling easy implementation of the phase-sensitive 2D NMR experiments. These methods also permit much higher receiver gains to be used to reduce digitization error, in particular, for the small resonances, which are sometimes vitally important for metabonomics studies. Both pulse sequences and experimental results are discussed for T(1)-, T(1rho)-, and T(2)-filtered COSY, T(2)-filtered phase-sensitive DQF-COSY, and T(1), T(1rho)-, and T(2)-filtered TOCSY NMR.     

Errors in RNA NOESY distance measurements in chimeric and hybrid duplexes: differences in RNA and DNA proton relaxation.

Nuclear magnetic resonance experiments reveal that the base H8/H6 protons of oligoribonucleotides (RNA) have T1 relaxation times that are distinctly longer than those of oligodeoxyribonucleotides (DNA). Similarly, the T1 values for the RNA H1' protons are approximately twice those of the corresponding DNA H1' protons. These relaxation differences persist in single duplexes containing covalently linked RNA and DNA segments and cause serious overestimation of distances involving RNA protons in typical NOESY spectra collected with a duty cycle of 2-3 s. NMR and circular dichroism experiments indicate that the segments of RNA maintain their A-form geometry even in the interior of DNA-RNA-DNA chimeric duplexes, suggesting that the relaxation times are correlated with the type of helix topology. The difference in local proton density is the major cause of the longer nonselective T1s of RNA compared to DNA, although small differences in internal motion cannot be completely ruled out. Fortunately, any internal motion differences that might exist are shown to be too small to affect cross-relaxation rates, and therefore reliable distance data can be obtained from time-dependent NOESY data sets provided an adequately long relaxation delay is used. In hybrid or chimeric RNA-DNA duplexes, if the longer RNA relaxation times are not taken into account in the recycle delay of NOESY pulse sequences, serious errors in measuring RNA proton distances are introduced.     

Nuclear magnetic resonance in environmental engineering: Principles and applications

This paper gives an introduction to nuclear magnetic resonance spectroscopy (NMR) and magnetic resonance imaging (MRI) in relation to applications in the field of environmental science and engineering. The underlying principles of high resolution solution and solid state NMR, relaxation time measurements and imaging are presented. Then, the use of NMR is illustrated and reviewed in studies of biodegradation and biotransformation of soluble and solid organic matter, removal of nutrients and xenobiotics, fate of heavy metal ions, and transport processes in bioreactor systems.     

Studies of individual carbon sites of proteins in solution by natural abundance carbon 13 nuclear magnetic resonance spectroscopy. Relaxation behavior.

The aromatic regions in proton-decoupled natural abundance 13C Fourier transform nuclear magnetic resonance spectra (at 14.2 kG) of small native proteins contain broad methine carbon bands and narrow nonprotonated carbon resonances. Some factors that affect the use of natural abundance 13C Fourier transform NMR spectroscopy for monitoring individual nonprotonated aromatic carbon sites of native proteins in solution are discussed. The effect of protein size is evaluated by comparing the 13C NMR spectra of horse heart ferrocytochrome c, hen egg white lysozyme, horse carbon monoxide myoglobin, and human adult carbon monoxide hemoglobin. Numerous single carbon resonances are observed in the aromatic regions of 13C NMR spectra of cytochrome c, lysozyme, and myoglobin. The much larger hemoglobin yields few resolved individual carbon resonances. Theoretical and some experimental values are presented for the natural linewidths (W), spin-lattice relaxation times (T1), and nuclear Overhauser enhancements (NOE) of nonprotonated aromatic carbons and Czeta of arginine residues. In general, the 13C-1H dipolar mechanism dominates the relaxation of these carbons. 13C-14N dipolar relaxation contributes significantly to 1/T1 of C epsilon2 of tryptophan residues and Czeta of arginine residues of proteins in D2O. The NOE of each nonprotonated aromatic carbon is within experimental error of the calculated value of about 1.2. As a result, integrated intensities can be used for making a carbon count. Theoretical results are presented for the effect of internal rotation on W, T1, and the NOE. A comparison with the experimental T1 and NOE values indicates that if there is internal rotation of aromatic amino acid side chains, it is not fast relative to the over-all rotational motion of the protein.     

23Na and flame photometric studies of the NMR visibility of sodium in rat muscle.

23Na nuclear magnetic resonance spectroscopy (NMR) is increasingly being used to study Na+ gradients and fluxes in biological tissues. However, the quantitative aspects of 23Na NMR applied to living systems remain controversial. This paper compares sodium concentrations determined by 23Na NMR in intact rat hindlimb (n = 8) and excised rat gastrocnemius muscle (n = 4) with those obtained by flame photometric methods. In both types of samples, 90% of the sodium measured by flame photometry was found to be NMR-visible. This is much higher than previously reported values. The NMR measurements for intact hindlimb correlated linearly with the flame photometric measurements, implying that one pool of sodium, predominantly extracellular, is 100% visible. From measurements on excised muscle, in which extracellular space is more clearly defined, the NMR visibility of intracellular Na+ was calculated to be 70%, assuming an extracellular space of 12% of the total tissue water volume and an extracellular NMR visibility of 100%. 23Na transverse relaxation measurements were carried out using a Hahn spin echo on both intact hindlimb (n = 1) and excised muscle (n = 2) samples. These showed relaxation curves that could each be described adequately using two relaxation times. The rapidly relaxing component showed a T2 value of 3-4 ms and the slowly relaxing component a T2 of 21-37 ms. A spin lattice relaxation (T1) measurement on intact hindlimb yielded a value of 51 ms. These relatively long relaxation times show that the quadrupolar relaxation effect of Na+ complexing to large macromolecules or being otherwise motionally restricted is relatively weak. This is consistent with the high NMR visibilities reported here.     

Viscosity of water in hibernating and nonhibernating mammals estimated by proton NMR relaxation times.

Longitudinal (T1) and transverse (T2) nuclear magnetic resonance relaxation times were measured in vitro at 37, 30, 25, 15, and 5 degrees C on serum, brain, liver, kidney, and heart samples from a hibernator, the European hamster, active in summer (SA), active in winter, or in the hibernating state in winter; from a less efficient hibernator, the golden hamster; and from a homeotherm, the rat. T1 and T2 relaxation times varied between species and in the European hamster between the active and hibernating subjects. Despite the major relaxation time differences between the organs, NMR relaxation time measurements showed a general trend to an increase in the viscosity of water for the European hamster in the active state. Although these modifications were not directly related to the process of hibernation itself, the relaxation times observed in the hibernating animals were closer to those seen in the rat. This evidenced that changes of physical properties of water reflect a better adaptation to low temperatures of the hamster, as compared to the nonhibernator, given that the low water viscosity of SA hamster allows the decrease of the viscosity with temperature during the hibernating state. These in vitro studies permit the study the viscosity which is an important physicochemical parameter involved in NMR longitudinal relaxation time of water proton. More detailed studies of other physiological parameters must be undertaken by further in vivo measurements.     

14N1H and 2H1H cross-relaxation in hydrated proteins.

The frequency dependence of the proton spin-lattice relaxation time T1 of solid hydrated bovine serum albumin and alpha-chymotrypsin has been measured over 4.5 decades in the range 10(4) to 3 X 10(8) Hz mainly by the aid of the field-cycling technique. The comparison between H2O- and D2O-hydrated samples permitted the distinction of exchangeable and unexchangeable protons. In all cases the 14N1H cross-relaxation dips due mainly to the amide groups have been observed. In addition, in the case of the deuterium exchanged proteins a 2H1H quadrupole dip appears. The amide groups act as relaxation sinks due to the coupling of the amide proton to 14N and adjacent protons. Outside of the dip regions the proton-proton coupling dominates. The fluctuations of the 14N1H and 1H1H interactions are of a different type. The unexchangeable protons show a T1 dispersion outside of the quadrupole dip regions given by the exceptional power law T1 approximately v0.75 +/- 0.05. It is shown that apart from structural information of the 14N spectra, 14N1H cross-relaxation spectroscopy permits the determination of correlation times in the range 10(-7) s less than tau less than 10(-4)S.     

Proton magnetic relaxation studies in normal and cancerous breast tissues

Proton magnetic resonance (PMR) relaxation times were measured for dissected malignant and normal tissue derived from breast cancer patients. Relaxation time measurements (T1, T2) were carried out at a RF frequency of 20 MHz and at a temperature of 27 degrees C with a Brucker PC 120 NMR Process analyser. The tissue types were confirmed by histopathological examination. In general T1 values were found to be longer for malignant tissues as compared to normal tissues which is in agreement with the earlier observations. The measured T2 values do not exhibit the malignant tissues above. The percentage of water content was also measured in both normal and malignant tissue and was found to be considerably larger in tumour tissue as compared to normal tissue. These results are discussed on the basis of two fraction fast exchange models of water molecules and confirm that PMR relaxation time measurement plays an important role in the differentiation of cancerous tissues from that of normal.     

Nuclear magnetic resonance study of muscle water protons in muscular dystrophy of chickens

Using the pulsed nuclear magnetic resonance (NMR) spectroscopy, the spin-lattice (T1) and the spin-spin (T2) relaxations times of water protons from samples of pectoralis major muscles of normal (line 412) and homozygous dystrophic (line 413) chickens were measured. Both the T1 and T2 were significantly increased (P less than 0.05) in the dystrophic muscles. The mean values of the relaxation times are given +/- S.D. The T1 values were 654 +/- 22 msec in normal and 692 +/- 41 msec in dystrophic muscles. The T2 values for normal and dystrophic muscles were 39 +/- 4 msec and 52 +/- 7 msec, respectively. Although the water content of dystrophic muscles (78.9 +/- 0.6%) determined by gravimetric methods was significantly higher than normal muscles (74.9 +/- 1.1%), this difference in tissue hydration could not explain quantitatively the increase of T1 and T2 values in the dystrophic muscles. The results of the measurements of the relaxation times seem to suggest that there are changes in the composition and/or conformational state of the proteins.     

Study of water and oil bodies in seeds by nuclear magnetic resonance

Two techniques of nuclear magnetic resonance (NMR) have been used for the study of water and lipids reserves in seeds. The temperature dependence of T1 relaxation time helps to identify differences in the thermodynamic properties of water between dry seeds and during germination. Among the species studied, T2 measurements distinguish two categories of seeds: pea, maize and wheat for which two components of T2 are observed, and lettuce, tomato and radish which present one single component. The main short component is attributed to water whereas the long one is attributed to lipids from oil bodies. Images of two dry seeds, one of pea and the other of radish, show marked differences in the distribution of NMR signal intensity, which suggest a different distribution of oil bodies.     

Molecular dynamics of the local anesthetic tetracaine in phospholipid vesicles

Upon introduction into phosphatidylcholine vesicles, the 13C magnetic resonance peaks of the aromatic resonances of tetracaine are broadened while the T1 relaxation times show little change. Addition of tetracaine to vesicles containing 30% cholesterol produces a similar broadening in the 13C NMR spectrum of tetracaine. Nuclear magnetic resonance parameters of phosphatidylcholine in vesicles which are unchanged by the addition of equimolar tetracaine include 13C T1 relaxation time and 31P linewidth, T1 relaxation time, and nuclear Overhauser effect enhancement. These results are interpreted as indicating a hydrophobic interaction between hydrocarbon portions of the anesthetic and phospholipid bilayer. The rotational correlation time of tetracaine about its long axis in the vesicles has been calculated from the 13C NMR spin lattice relaxation times to be about 10(-10.3) s and is unchanged by incorporation into the phospholipid bilayer. The positively charged ammonium group of tetracaine interacts with the negatively charged phosphate group of the vesicle lipids. Using shift reagents and 31P NMR, tetracaine has been shown to displace cations from the bilayer surface, and does not undergo fast flip-flop across the vesicle bilayer.     

19F NMR investigation of molecular motion and packing in sonicated phospholipid vesicles

Dimyristoylphosphatidylcholine (DMPC) labeled with a C19F2 group in the 4-, 8-, or 12-position of the 2-acyl chain has been investigated in sonicated unilamellar vesicles (SUV) by fluorine-19 nuclear magnetic resonance (NMR) at 282.4 MHz from 26 to 42 degrees C. The 19F NMR spectra exhibit two overlapping resonances with different line widths. Spin-lattice relaxation time measurements have been performed in both the laboratory frame (T1) and the rotating frame (T1 rho) in order to investigate the packing and dynamics of phospholipids in lipid bilayers. Quantitative line-shape and relaxation analyses are possible by using the experimental chemical shift anisotropy (delta nu CSA) and the internuclear F-F vector order parameter (SFF) values obtained from the 19F powder spectra of multilamellar liposomes. The following conclusions can be made: The 19F chemical shift difference between the inside and outside leaflets of SUV can be used to monitor the lateral packing of the phospholipid in the two SUV monolayers. The hydrocarbon chains in the outer layer are found to be more tightly packed than those of the inner one, and the differences between them become smaller near the chain terminals. The effective correlation time [(1-4) x 10(-7) s] obtained from either the motional narrowing of the line widths or off-resonance T1 rho measurements is shorter than that estimated from the Stokes-Einstein diffusion model (10(-6) s), on the basis of a hydrodynamic radius of 110 A for SUV.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intermolecular interactions between the SH3 domain and the proline-rich TH region of Bruton's tyrosine kinase

The SH3 domain of Bruton's tyrosine kinase (Btk) is preceded by the Tec homology (TH) region containing proline-rich sequences. We have studied a protein fragment containing both the Btk SH3 domain and the proline-rich sequences of the TH region (PRR-SH3). Intermolecular NMR cross-relaxation measurements, gel permeation chromatography profiles, titrations with proline-rich peptides, and (15)N NMR relaxation measurements are all consistent with a monomer-dimer equilibrium with a dissociation constant on the order of 60 microM. The intermolecular interactions do, at least in part, involve proline-rich sequences in the TH region. This behavior of Btk PRR-SH3 may have implications for the functional action of Btk.     

Structural studies on membrane proteins using non-linear spin label EPR spectroscopy

Non-linear electron spin resonance (EPR) techniques suitable for measuring proximity relationships in membranes are reviewed. These were developed during the past decade in order to measure changes sensitively in the spin-lattice relaxation time (T1) of nitroxyl spin labels covalently attached to membrane lipids or proteins. In combination with paramagnetic quenching agents and double spin-labelling, the methods were further developed for distance measurements. Selected examples are given to illustrate different methods, and types of data obtained for both integral and peripheral membrane proteins.     

Water proton magnetic resonance studies of normal and sickle erythrocytes. Temperature and volume dependence.

The temperature and cell volume dependence of the NMR water proton line-width, spin-lattice, and spin-spin relaxation times have been studied for normal and sickle erythrocytes as well as hemoglobin A and hemoglobin S solutions. Upon deoxygenation, the spin-spin relaxation time (T2) decreases by a factor of 2 for sickle cells and hemoglobin S solutions but remains relatively constant for normal cells and hemoglobin A solutions. The spin-lattice relaxation time (T1) shows no significant change upon deoxygenation for normal or sickle packed red cells. Studies of the change in the NMR linewidth, T1 and T2 as the cell hydration is changed indicate that these parameters are affected only slightly by a 10-20% cell dehydration. This result suggests that the reported 10% cell dehydration observed with sickling is not important in the altered NMR properties. Low temperature studies of the linewidth and T1 for oxy and deoxy hemoglobin A and hemoglobin S solutions suggest that the "bound" water possesses similar properties for all four species. The low temperature linewidth ranges from about 250 Hz at -15 degrees C to 500 Hz at -36 degrees C and analysis of the NMR curves yield hydration values near 0.4 g water/g hemoglobin for all four species. The low temperature T1 data go through a minimum at -35 degrees C for measurements at 44.4 MHz and -50 degrees C for measurements at 17.1 MHz and are similar for oxy and deoxy hemoglobin A and hemoglobin S. These similarities in the low temperature NMR data for oxy and deoxy hemoglobin A and hemoglobin S suggest a hydrophobically driven sickling mechanism. The room temperature and low temperature relaxation time data for normal and sickle cells are interpreted in terms of a three-state model for intracellular water. In the context of this model the relaxation time data imply that type III, or irrotationally bound water, is altered during the sickling process.