Line shape analyses for water 17O NMR quintet observed in a bacteriophage Pf1 solution at different temperatures

Partially resolved 17O NMR quintet was observed in a filamentous bacteriophage Pf1 solution at 70 degrees C with a quadrupole splitting approximately 100 Hz. As the temperature decreased, the resolution was reduced but the line shapes were still indicative of residual quadrupole splitting. Line shape analyses were performed using the quadrupolar relaxation theory for spin 5/2. The contribution to the residual quadrupole splitting from the electric field gradients stemming from the phage filaments, which were oriented in the magnet, was taken into account. As a result, the observed 17O spectra at different temperatures were simulated and the hydration number of the phage DNA was determined.     

Sodium interaction with ordered structures in mammalian red blood cells detected by Na-23 double quantum NMR.

Na-23 double and triple quantum filtered NMR spectra of intact dog and human red blood cells were measured with the pulse sequence 90 degrees-tau/2-180 degrees-tau/2-theta degrees-t1-theta degrees-t2(Acq). For theta = 90 degrees the triple quantum filtered spectra exhibited the typical multiple quantum filtered lineshape, characteristic of isotropic media, while the double quantum filtered ones presented a superposition of two signals, whose proportion depended on the creation time tau. This effect is due to the formation of both second and third rank tensors. The formation of the second rank tensor, T21 results from non-zero residual quadrupolar interaction and is related to the anisotropic motion of sodium ions. Measurements of the double quantum filtered spectra with theta = 54.7 degrees enabled the detection of the contribution of T21 exclusively. No residual quadrupolar interaction was detected for sodium in the cytoplasm, while unsealed ghosts displayed the double quantum filtered spectral pattern, similar to that of intact cells. The anisotropy of motion of the sodium at the plasma membrane of mammalian erythrocytes depended on the integrity of the cytoskeleton network. Theoretical analysis of the double quantum filtered spectra gave a value of residual quadrupolar splitting of approximately 20 Hz for intact unsealed ghosts. The data presented prove that double quantum filtering is a sensitive technique for detection of motional anisotropies in biological systems.     

The source of NMR-detected motional anisotropy of water in blood vessel walls.

2H Double quantum-filtered (DQF) NMR spectroscopy of deuterated water is sensitive to the presence of order in biological systems. This is because the only nuclei that are detected are those with residual quadrupolar interactions due to their anisotropic motion. In the present study, samples of aorta, coronary and carotid arteries, and vena cava were studied in parallel by 2H DQF NMR and by light microscopy. The average quadrupolar splitting, calculated from the NMR data, varies considerably among the different blood vessels, with high reproducibility for each type of vessel. Polarization microscopy examinations using collagen-specific staining with picrosirius red, have shown a variety of color profiles for the different blood vessels. These reflect different physical modes of aggregation (packing and thickness) of collagen fibers. A correlation was found between the NMR parameters and the color profiles of the picrosirius red-stained sections. Treating the blood vessels with 90% formic acid resulted in the elimination of the 2H DQF NMR signal. Histological analysis demonstrated a complete degradation of collagen and muscle, whereas the elastin filaments were preserved. Evidence is given that the 2H DQF NMR signal is dominated by the contribution of water molecules interacting with the collagen fibers.     

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.     

Deuterium NMR investigation of an amphotericin B derivative in mechanically aligned lipid bilayers

The methyl-d(3) amide derivative of the polyene antibiotic amphotericin B was synthesized, assayed for biological activity, incorporated into mechanically aligned bilayers of dipalmitoylphosphatidylcholine (DPPC), and examined by deuterium and phosphorus NMR. The amide derivative has a lesser, but qualitatively similar, biological activity relative to amphotericin B. Incorporation of the amide derivative and ergosterol into aligned DPPC bilayers resulted in a single, stable bilayer phase, as shown by phosphorus NMR of the DPPC headgroups. Deuterium NMR spectra revealed one major (2)H quadrupolar splitting and one major (2)H-(1)H dipolar splitting in the liquid-crystalline phase, consistent with a high degree of alignment and a single, averaged physical state for amphotericin B methyl-d(3) amide in the bilayer. Variations of the quadrupolar and dipolar splittings as a function of macroscopic sample orientation and temperature indicated that the amide derivative undergoes fast rotation about a motional axis that is parallel to the bilayer normal.     

An NMR study of pyridine associated with DMPC liposomes and magnetically ordered DMPC-surfactant mixed micelles.

With molecular dynamics simulations of phospholipid membranes becoming a reality, there is a growing need for experiments that provide the molecular details necessary to test these computational results. Pyridine is used here to explore the interaction of planar aromatic groups with the water-lipid interface of membranes. It is shown by magic angle spinning 13C nuclear magnetic resonance (NMR) to bind between the glycerol and choline groups of dimyristoylphosphatidylcholine (DMPC) liposomes. The axial pattern for the 31P NMR spectrum of DMPC liposomes is preserved even with more than half of the interfacial sites occupied, indicating that pyridine does not disrupt the lamellar phase of this lipid. 2H NMR experiments of liposomes in deuterium oxide demonstrate that pyridine might promote greater penetration of water into restricted regions in the interface. Magnetically oriented DMPC/surfactant micelles were investigated as a means for improving resolution and sensitivity in NMR studies of species bound to bilayers. The quadrupolar splittings in the 2H NMR spectra of d5-pyridine in DMPC liposomes and magnetically oriented DMPC/Trixon X-100 micelles indicate a common bound state for the two bilayer systems. The well resolved quadrupolar splittings of d5-pyridine in oriented micelles were used to establish the tilt of the pyridine ring relative to the bilayer plane.     

Deuterium nuclear magnetic resonance investigation of the exchangeable sites on gramicidin A and gramicidin S in multilamellar vesicles of dipalmitoylphosphatidylcholine

Solid gramicidin A and S and their interaction with DPPC bilayers were examined by 2H NMR as well as 31P NMR and differential scanning calorimetry (DSC). The deuterium spectra arose from deuterons associated with the peptide through chemical exchange in 2H2O. The spectra from both peptides were characterized by a quadrupolar splitting parameter, omega Q/2 pi approximately 150 kHz, and an asymmetry parameter, eta approximately 0.17. An additional 33 kHz, eta = 0 component arising from deuterons on mobile ornithine side chains was present in gramicidin S. In the gel phase of dipalmitoylphosphatidylcholine liposomes the gramicidins gave spectra that had components identical with those obtained from the solids. In the liquid-crystalline phase gramicidin A containing samples gave multicomponent spectra with a maximum quadrupolar splitting value of 133 kHz, eta = 0. A minimum in the T2e was observed, coinciding with the onset of the broadened phase transition measured by DSC and 31P NMR, due to the onset of axial rotation of the peptide in the bilayer. The different powder patterns in the liquid-crystalline spectra from gramicidin A probably arise from different amide sites along the transmembrane channel. The broad component of the 2H NMR spectra from gramicidin S in liposome preparations was not affected by the lipid-phase transition. The T2e was also constant over this temperature range. The results are consistent with a location of gramicidin S at the membrane surface.     

Oxygen-17 and deuterium nuclear magnetic relaxation studies of lysozyme hydration in solution: field dispersion, concentration, pH/pD, and protein activity dependences

A comparison of 17O and 2H NMR relaxation rates of water in lysozyme solutions as a function of concentration, pH/pD, and magnetic field suggests that only 17O monitors directly the hydration of lysozyme in solution. NMR measurements are for the first time extended to 11.75 T. Lysozyme hydration data are analyzed in terms of an anisotropic, dual-motion model with fast exchange of water between the "bound" and "free" states. The analysis yields 180 mol "bound" water/mol lysozyme and two correlation times of 7.4 ns ("slow") and 29 ps ("fast") for the bound water population at 27 degrees C and pH 5.1, in the absence of salt, assuming anisotropic motions of water with an order parameter value for bound water of 0.12. Under these conditions, the value of the slow correlation time of bound water (7.4 ns) is consistent with the value of 8 ns obtained by frequency-domain fluorescence techniques for the correlation time associated with the lysozyme tumbling motion in solutions without salt. In the presence of 0.1 M NaCl the hydration number increases to 290 mol/mol lysozyme at pD 4.5 and 21 degrees C. The associated correlation times at 21 degrees C in the presence of 0.1 M NaCl are 4.7 ns and 15.5 ps, respectively. The value of the slow correlation time of 4.7 ns is consistent with the calculated value (4.9 ns) for the lysozyme monomer tumbling in solution. The systematic deviations of the relaxation rates, estimated with the single-exponential approximation, from the theoretical, multiexponential nuclear (I' + 1/2) spin relaxation are evaluated at various frequencies for 17O (I = 5/2) with the first-order, linear approximation (25). All NMR relaxation data for hydrated lysozymes are affected by protein activity and are sensitive both to the ionization of protein side chains and to the state of protein aggregation.     

Deuterium solid-state NMR investigations of exchange labeled oriented purple membranes at different hydration levels

Oriented purple membranes were equilibrated under controlled (2)H(2)O relative humidity ranging from 15% to 93% and introduced into the magnetic field of an NMR spectrometer with the membrane normal parallel to the magnetic field direction. Deuterium solid-state NMR spectra of these samples resolved four deuteron populations. Deuterons that have exchanged with amide protons of the protein exhibited a broad spectral line shape (<150 kHz). Furthermore, a broadened signal of deuterons tightly associated with protein and lipid is detected at low hydration, as well as two additional water populations that were present when the samples were equilibrated at >/=75% relative humidity. These latter ones are characterized by narrow quadrupolar splittings (<2.5 kHz) and orientation-dependent chemical shifts. Their deuterium relaxation times, measured as a function of temperature, indicate correlation times in the fast regime (10(-10) s) and activation energies of 13 kJ/mol (at 86% relative humidity). Differences in T(1) and T(2) relaxation together with small residual quadrupole splittings show that the mobility of the deuterons is anisotropic. The occurrence of these mobile water populations at high levels of purple membrane hydration (>/=75% relative humidity) correlate with proton pumping activity of bacteriorhodopsin, the fast kinetics of M-decay in the bacteriorhodopsin photocycle, and structural alterations of the protein during the M-state, which have been described previously.     

<Superscript>2</Superscript>H–<Superscript>13</Superscript>C correlation solid-state NMR for investigating dynamics and water accessibilities of proteins and carbohydrates

Site-specific determination of molecular motion and water accessibility by indirect detection of ²H NMR spectra has advantages over dipolar-coupling based techniques due to the large quadrupolar couplings and the ensuing high angular resolution. Recently, a Rotor Echo Short Pulse IRrAdiaTION mediated cross polarization (^RESPIRATIONCP) technique was developed, which allowed efficient transfer of ²H magnetization to ¹³C at moderate ²H radiofrequency field strengths available on most commercial MAS probes. In this work, we investigate the ²H–¹³C magnetization transfer characteristics of one-bond perdeuterated CD _n spin systems and two-bond H/D exchanged C–(O)–D and C–(N)–D spin systems in carbohydrates and proteins. Our results show that multi-bond, broadband ²H–¹³C polarization transfer can be achieved using ²H radiofrequency fields of ~50 kHz, relatively short contact times of 1.3–1.7 ms, and with sufficiently high sensitivity to enable 2D ²H–¹³C correlation experiments with undistorted ²H spectra in the indirect dimension. To demonstrate the utility of this ²H–¹³C technique for studying molecular motion, we show ²H–¹³C correlation spectra of perdeuterated bacterial cellulose, whose surface glucan chains exhibit a motionally averaged C6 ²H quadrupolar coupling that indicates fast trans-gauche isomerization about the C5–C6 bond. In comparison, the interior chains in the microfibril core are fully immobilized. Application of the ²H–¹³C correlation experiment to H/D exchanged Arabidopsis primary cell walls show that the O–D quadrupolar spectra of the highest polysaccharide peaks can be fit to a two-component model, in which 74% of the spectral intensity, assigned to cellulose, has a near-rigid-limit coupling, while 26% of the intensity, assigned to matrix polysaccharides, has a weakened coupling of 50 kHz. The latter O–D quadrupolar order parameter of 0.22 is significantly smaller than previously reported C–D dipolar order parameters of 0.46–0.55 for pectins, suggesting that additional motions exist at the C–O bonds in the wall polysaccharides. ²H–¹³C polarization transfer profiles are also compared between statistically deuterated and H/D exchanged GB1.     

The mitochondrial precursor protein apocytochrome c strongly influences the order of the headgroup and acyl chains of phosphatidylserine dispersions. A 2H and 31P NMR study

Deuterium and phosphorus nuclear magnetic resonance techniques were used to study the interaction of the mitochondrial precursor protein apocytochrome c with headgroup-deuterated (dioleoylphosphatidyl-L-[2-2H1]serine) and acyl chain deuterated (1,2-[11,11-2H2]dioleoylphosphatidylserine) dispersions. Binding of the protein to dioleoylphosphatidylserine liposomes results in phosphorus nuclear magnetic resonance spectra typical of phospholipids undergoing fast axial rotation in extended liquid-crystalline bilayers with a reduced residual chemical shift anisotropy and an increased line width. 2H NMR spectra on headgroup-deuterated dioleoylphosphatidylserine dispersions showed a decrease in quadrupolar splitting and a broadening of the signal on interaction with apocytochrome c. Addition of increasing amounts of apocytochrome c to the acyl chain deuterated dioleoylphosphatidylserine dispersions results in the gradual appearance of a second component in the spectra with a 44% reduced quadrupolar splitting. Such large reduction of the quadrupolar splitting has never been observed for any protein studied yet. The lipid structures corresponding to these two components could be separated by sucrose gradient centrifugation, demonstrating the existence of two macroscopic phases. In mixtures of phosphatidylserine and phosphatidylcholine similar effects are observed. The induction of a new spectral component with a well-defined reduced quadrupolar splitting seems to be confined to the N-terminus since addition of a small hydrophilic amino-terminal peptide (residues 1-38) also induces a second component with a strongly reduced quadrupolar splitting. A chemically synthesized peptide corresponding to amino acid residues 2-17 of the presequence of the mitochondrial protein cytochrome oxidase subunit IV also has a large perturbing effect on the order of the acyl chains, indicating that the observed effects may be a property shared by many mitochondrial precursor proteins. In contrast, binding of the mature protein, cytochrome c, to acyl chain deuterated phosphatidylserine dispersions has no effect on the deuterium and phosphorus nuclear magnetic resonance spectra, thereby demonstrating precursor-specific perturbation of the phospholipid order. The inability of holocytochrome c to perturb the phospholipid order is due to folding of this protein, since unfolding of cytochrome c by heat or urea treatment results in similar effects on dioleoylphosphatidylserine bilayers, as observed for the unfolded precursor. Implications of these data for the import of apocytochrome c into mitochondria will be discussed.     

Binding of 17O-labeled substrate and inhibitors to protocatechuate 4,5-dioxygenase-nitrosyl complex. Evidence for direct substrate binding to the active site Fe2+ of extradiol dioxygenases

Pseudomonas testosteroni protocatechuate 4,5-dioxygenase catalyzes extradiol-type oxygenolytic cleavage of the aromatic ring of its substrate. The essential active site Fe2+ binds nitric oxide (NO) to produce an EPR active complex with an electronic spin of S = 3/2. Hyperfine broadening of the EPR resonances of the nitrosyl complex of the enzyme by protocatechuate (3,4-(OH)2-benzoate, PCA) enriched specifically with 17O (I = 5/2) in either the 3 or the 4 hydroxyl group shows that both groups can bind directly to the Fe2+ in the ternary complex. Analogous results are obtained for PCA binding to catechol 2,3-dioxygenase-NO complex suggesting that substrate binding by the Fe2+ may be a general property of extradiol dioxygenases. The protocatechuate 4,5-dioxygenase inhibitor, 4-17OH-benzoate binds directly to the Fe of the nitrosyl adduct of the enzyme through the OH group. Since previous studies have shown that water also is bound to the Fe in this ternary complex, but not in the ternary complex with PCA, the data strongly imply that there are 3 sites in the Fe coordination which can be occupied by exogenous ligands. 3-17OH-benzoate is an inhibitor of the enzyme but does not elicit detectable hyperfine broadening in the EPR spectrum of the nitrosyl adduct suggesting that it binds to the enzyme, but not to the Fe. The EPR spectra of ternary enzyme-NO complexes with PCA or 4-OH-benzoate labeled with 17O exclusively in the carboxylate substituent are not broadened, suggesting that this moiety does not bind to the Fe.     

Orientational ordering and dynamics of the hydrate and exchangeable hydrogen atoms in crystalline crambin

Deuterium nuclear magnetic resonance studies of crambin crystals grown from deuterated solvent (2H2O/CH3CH2O2H or H2O/C2H3CH2OH) are reported. The extent to which the hydrate and exchangeable hydrogen atoms are dynamically disordered are then determined from the size of the residual deuterium quadrupole couplings, qcc. Rapid molecular reorientation (tau c-1 greater than 10(5) s-1) reduces the magnitude of the quadrupole coupling from its static value (216 kHz for solid water). We find that the room temperature spectrum of crambin is dominated by two features: a sharp line with very small residual quadrupolar coupling less than 3 kHz, and a broad pattern with a quadrupolar coupling in the range 185 to 195 kHz. The former is indicative of very nearly isotropically reorienting deuterons, whereas the latter is somewhat narrower than that observed for the amide deuterons of poly-gamma-benzyl-L-glutamate and thus indicative of deuterons that are almost but not completely stationary. By considering the nuclear magnetic resonance spectrum intensities along with the amino acid sequence, X-ray structure and the manner in which quadrupole couplings are reduced by dynamics, we conclude that the nuclear magnetic resonance signal from most of the water molecules of hydration are contained in the sharp line, i.e. reorient nearly isotropically in the crystalline protein. Unlike bulk water, which freezes abruptly in the manner of a phase transition, the water of hydration in crambin has a broad freezing range from 180 to 250K, as evidenced by the decreasing intensity of the sharp line that disappears at 180K. At temperatures between 150 and 200K, a typical hydrate molecule reorients at a rate comparable to the quadrupole coupling, 10(4) s-1 to 10(5) s-1, a process that occurs in hexagonal ice in the range of 240 to 270K. At 140K, the hydrate is stationary, tau c-1 less than 10(3) s-1. Studies of the protein crystallized from solvent deuterated only at the non-exchangeable methyl group of ethanol confirm that ethanol is in the lattice and show that this solvate behaves in much the same way as the hydrate. The refined X-ray structure has identified four ethanol solvate molecules. The deuterium spectrum at room temperature has a well-defined residual pattern with qcc = 2.2 kHz, i.e. a small-order parameter consistent with nearly isotropically reorienting molecules. The spectrum width broadens substantially only at temperatures below 200K and achieves the characteristic spectrum of a rotating methyl group with stationary C-C axis at 140K.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthesis of (Rp,Rp)-P1,P4-Bis(5'-adenosyl)-1[17O,18O2],4[17O,18O2] tetraphosphate from (Sp,Sp)-P1,P4-Bis(5'-adenosyl)-1[thio-18O2], 4[thio-18O2]tetraphosphate with retention at phosphorus and the stereochemical course of hydrolysis by the unsymmetrical Ap4A phosphodiesterase from lupin seeds

The three stereoisomers of P1,P4-bis(5'-adenosyl)-1,4-dithiotetraphosphate have been synthesized and their 31P NMR spectra investigated. The effect of temperature on the circular dichroic spectrum of the (Sp,Sp)-stereoisomer shows that unstacking of the molecule occurs as the temperature is raised. Treatment of the (Sp,Sp)-stereoisomer with cyanogen bromide in [18O]water leads to substitution of sulfur by 18O with predominant retention of configuration at P1 and P4. (Sp,Sp)-P1,P4-Bis(5'-adenosyl)-1[thio-18O2],4[thio-18O2]tetraphosphate was synthesized and on treatment with cyanogen bromide in [17O]water gave (Rp,Rp)-P1,P4-bis(5'-adenosyl)-1[17O,18O2],4[17O,18O2]tetraphosphate. Hydrolysis by unsymmetrical Ap4A phosphodiesterase from lupin seeds gave (Rp)-5'-[16O,17O,18O]AMP. The reaction therefore proceeds with inversion of configuration at phosphorus, indicating that the enzyme-catalyzed displacement by water occurs by a direct "in-line" mechanism.     

2H nuclear magnetic resonance of exchange-labeled gramicidin in an oriented lyotropic nematic phase

Lyotropic nematic liquid-crystalline phases, such as that formed by potassium laurate/decanol/KCl/water, are found to accept readily large amphiphilic solute molecules. Since these phases spontaneously orient in high magnetic fields, it becomes possible to obtain NMR spectra of biologically interesting solutes in an oriented axially symmetric environment. The amide hydrogens of the peptide backbone of gramicidin D (Dubos) were exchanged for deuterium, and the gramicidin was incorporated into a lyotropic nematic phase made with deuteriated buffer in place of water. 2H NMR spectra of oriented, exchange-labeled gramicidin were then obtained. The strong water signal from the deuteriated buffer was eliminated by using selective excitation and a polynomial subtraction procedure. The 2H NMR spectra at high temperature consist of twelve major quadrupolar doublets. The splittings observed are largely independent of temperature, suggesting a highly rigid backbone structure. Two of the doublets, which are chemically shifted relative to the others, show stronger temperature dependence. These two probably arise from the exchangeable amino hydrogens on the tryptophan indole moieties of the peptide. While we cannot yet assign all of the doublets, the spectra and nuclear magnetic relaxation data are consistent with a rigid slightly distorted beta LD6.3 helix undergoing axially symmetric reorientation about the director of the liquid-crystalline phase. The correlation time for the axially symmetric reorientation is determined by relaxation measurements to be about 10(-7) s.     

Multinuclear NMR investigation of the NaDNA/ethidium bromide anisotropic system

A combined use of (31)P, (23)Na, (2)H and (17)O NMR spectroscopies and polarized light microscopy has been employed to investigate the effect of the ethidium bromide (EB) binding on the liquid crystalline phase of concentrated double stranded DNA solutions. The optical textures and the (31)P and (23)Na NMR spectra of the DNA anisotropic solutions show that the intercalation of EB induces significant modifications either in the arrangements of the DNA rods and the surrounding ionic atmosphere. On the contrary, no indication of significant changes of the orientational order of the water molecules around DNA emerges from the water (2)H and (17)O NMR spectra.     

Polyethylene glycol as a hydration agent in oriented membrane bilayer samples

Techniques such as NMR, ESR, fluorescence depolarization, and neutron scattering are commonly used to investigate the physical properties of membranes. Oriented membrane bilayer systems (single crystals) are often employed in these investigations. It is important to know and be able to control the level of hydration in these samples. In particular, one must have confidence that a sample is in fact “fully hydrated” and remains so during the course of the experiment. Full hydration is difficult to obtain by hydrating oriented samples using water-saturated vapor. An alternative method for hydrating oriented samples is to surround the oriented sample by a polymer solution. Higher hydration levels are achieved using this method. Three nuclear magnetic resonance studies using headgroup deuterated 1,2-dipalmitoyl-sn-glycerol-3-phosphocholine (DPPC) were done to compare the hydration level of oriented headgroup samples surrounded by a polymer/water solution and fully hydrated multibilayer dispersions. Transition temperatures, quadrupolar splittings (at 50°C) and spin-lattice relaxation times (at 50°C) were measured. The simple tests of the transition temperature and quadrupolar splitting to determine full hydration, as my results show, are not sufficient. In this paper I demonstrate that more fully hydrated samples can easily be achieved by surrounding the oriented sample with a 5 wt% polyethylene glycol/water solution than by hydrating in water saturated vapor.     

Examination of structural characteristics of the potent oxytocin antagonists [dPen1,Pen6]-OT and [dPen1,Pen6, 5-tBuPro7]-OT by NMR, Raman, CD spectroscopy and molecular modeling.

The synthesis and biological evaluation of penicillamine(6)-5-tert-butylproline(7)-oxytocin analogs and comparison with their proline(7)-oxytocin counterparts has led to the discovery of two potent oxytocin (OT) antagonists: [dPen(1),Pen(6)]-oxytocin (1, pA(2) = 8.22, EC(50) = 6.0 nM) and [dPen(1),Pen(6),5-tBuPro(7)]-oxytocin (2, pA(2) = 8.19, EC(50) = 6.5 nM). In an attempt to understand the conformational requirements for their biological activity, spectroscopic analyses of 1 and 2 were performed using (1)H NMR, laser Raman and CD techniques. In H(2)O, oxytocin analogs 1 and 2 exhibited cis-isomer populations of 7% and 35%, respectively. Measurement of the amide proton temperature coefficients revealed solvent shielded hydrogens for Gln(4) and Pen(6) in the major trans-conformer of 1 as well as for Gln(4) in the minor cis-conformer of 2. Few long-distance NOEs were observed, suggesting conformational averaging for analogs 1 and 2 in water; moreover, a lower barrier (16.6 +/- 0.2 kcal/mol) for isomerization of the amide N-terminal to 5-tBuPro(7) relative to OT was calculated from measuring the coalescence temperature of the Gly(9) backbone NH signals in the NMR spectra of 2. Observed bands in the Raman spectra of 1 and 2 correspond to C(beta)-S-S-C(beta) dihedral angles of +110-115 degrees and +/-90 degrees , respectively. In water, acetonitrile and methanol, the CD spectra for 1 exhibited a positive maximum around 236-239 nm; in trifluoroethanol, the spectra shifted and a negative maximum was observed at 240 nm. The CD spectra of 2 were unaffected by solvent changes and exhibited a negative maximum at 236-239 nm. The CD and Raman data both suggested that a conformation having a right-handed screw sense about the disulfide and a chi(CS-SC) dihedral angle value close to 115 degrees was favored for analog 1 in water, methanol and acetonitrile, but not trifluoroethanol, where a +/-90 degrees angle was favored. Analog 2 was more resilient to conformational change about the disulfide, and adopted a preferred disulfide geometry corresponding to a +/-90 degrees chi(CS-SC) dihedral angle. Monte Carlo conformational analysis of analogs 1 and 2 using distance restraints derived from NMR spectroscopy revealed two prominent conformational minima for analog 1 with disulfide geometries around +114 degrees and +116 degrees . Similar analysis of analog 2 revealed one conformational minimum with a disulfide geometry around +104 degrees . In sum, the conformation about the disulfide in [dPen(1),Pen(6)]-OT (1) was shown to be contingent on environment and in TFE, adopted a geometry similar to that of [dPen(1),Pen(6),5-tBuPro(7)]-OT (2) which appeared to be stabilized by hydrophobic interactions between the 5-tBuPro(7) (5R)-tert-butyl group, the Leu(8) isopropyl sidechain and the Pen(6)beta-methyl substituents. In light of the conformational rigidity of 2 about the disulfide bond, and the similar geometry adopted by 1 in TFE, a S-S dihedral angle close to +110 degrees may be a prerequisite for their binding at the receptor.     

Characterization of polyacrylamide-stabilized Pf1 phage liquid crystals for protein NMR spectroscopy

A new polymer-stabilized nematic liquid crystal has been characterized for the measurement of biomolecular residual dipolar couplings. Filamentous Pf1 phage were embedded in a polyacrylamide matrix that fixes the orientation of the particles. The alignment was characterized by the quadrupolar splitting of the ²H NMR water signal and by the measurement of ¹H-¹⁵N residual dipolar couplings (RDC) in the archeal translation elongation factor 1. Protein dissolved in the polymer-stabilized medium orients quantitatively as in media without polyacrylamide. We show that the quadrupolar splitting and RDCs are zero in media in which the Pf1 phage particles are aligned at the magic angle. This allows measurement of J and dipolar couplings in a single sample.     

The Nature and Origin of Chemical Shift for Intracellular Water Nuclei in Artemia Cysts

We investigated the possible existence of chemical shift of water nuclei in Artemia cysts using high resolution nuclear magnetic resonance (NMR) methods. The results conducted at 60, 200, and 500 MHz revealed an unusually large chemical shift for intracellular water protons. After correcting for bulk susceptibility effects, a residual downfield chemical shift of 0.11 ppm was observed in fully hydrated cysts. Similar results have been observed for the deuterium and ¹⁷O nuclei.

We have ruled out unusual intracellular pH, diamagnetic susceptibility of intracellular water, or interaction of water molecules with lipids, glycerol, and/or trehalose as possible origins of the residual chemical shift. We conclude that the residual chemical shift observed for water nuclei (¹H, ²H, and ¹⁷O) is due to significant water-macromolecular interactions.