2Q NMR of (2)H2O ordering at solid interfaces.

Solvent ordering at an interface can be studied by multiple-quantum NMR. Quantitative studies of (2)H2O ordering require clean double-quantum (2Q) filtration and an analysis of 2Q buildup curves that accounts for relaxation and, if randomly oriented samples are used, the distribution of residual couplings. A pulse sequence with absorption mode detection is extended for separating coherences by order and measuring relaxation times such as the 2Q filtered T2. Coherence separation is used to verify 2Q filtration and the 2Q filtered T2 is required to extract the coupling from the 2Q buildup curve when it is unresolved. With our analysis, the coupling extracted from the buildup curve in (2)H2O hydrated collagen was equivalent to the resolved coupling measured in the usual 1D experiment and the 2Q to 1Q signal ratio was in accord with theory. Application to buildup curves from (2)H2O hydrated elastin, which has an unresolved coupling, revealed a large increase in the 2Q signal upon mechanical stretch that is due to an increase in the ordered water fraction while changes in the residual coupling and T2 are small.

Synthesis and physico-chemical properties of peptides in soil humic substances.

Soil humic substances (HS) are heterologous, polydispersive, and multi-functional organometallic macromolecules ubiquitous in soils and sediments. They are key players in the maintenance of the belowground ecosystems and in the bioavailability of both organic and inorganic contaminants. It is widely assumed that the peptidic substructures of HS are readily degraded and therefore do not contribute significantly to interactions with contaminants such as toxic metals. To investigate the turnover of humified peptides, laboratory soil aging experiments were conducted with 13C-glucose or 15N-nitrate for 8.5 months. Evidence for random-coil peptidic structures in the labeled HS was obtained from 2-D nuclear magnetic resonance (NMR), pyrolysis gas chromatography-mass spectrometry (pyro-GC-MS), and circular dichroism data. Interaction of metals with the peptidic carbonyls of labeled HS was rationalized from the solid-state NMR data. Detailed 13C and 15N labeling patterns of amino acid residues in the acid hydrolysates of HS acquired from NMR and GC-MS revealed two pools of peptides, i.e. one extant (unlabeled) and the other, newly humified with little isotopic scrambling (fully labeled). The persistence of pre-existing peptidic structures indicates their resistance to degradation while the presence of fully labeled peptidic amino acids suggests wholesale incorporation of newly synthesized peptides into HS. These findings are contrary to the general notion that humified peptides are readily degraded.

Determination of the chemical shielding tensor orientation from two or one of the three conventional rotations of a single crystal.

Chemical shift anisotropy (CSA) is an immensely useful interaction to study the structure, dynamics, and function of a wide variety of chemical and biological molecules. Traditionally the only unambiguous way to determine both the principal values and the orientation of the principal axes of the CSA tensor has been to follow the chemical shift frequency changes as a crystal of known structure is rotated relative to the direction of the external magnetic field. This classic method employs rotations about three mutually orthogonal axes of a single crystal. It is shown here that just two, or one, of the above rotations suffice to determine the CSA tensor orientation by borrowing, the easy to obtain, principal values of CSA from an independent source. Methods for using two rotation patterns or even a single rotation pattern are described and illustrated with known chemical shielding tensors.

Backbone motions in a crystalline protein from field-dependent 2H-NMR relaxation and line-shape analysis.

We have used 2H-nmr to study backbone dynamics of the 2H-labeled, slowly exchanging amide sites of fully hydrated, crystalline hen egg white lysozyme. Order parameters are determined from the residual quadrupole coupling and values increase from S2 = 0.85 at 290 K to S2 = 0.94 at 200 K. Dynamical rates are determined from spin-lattice relaxation at three nmr frequencies (38.8, 61.5, and 76.7 MHz). The approach used here is thus distinct from solution nmr studies where dynamical amplitudes and rates are both determined from relaxation measurements. At temperatures below 250 K, relaxation is independent of the nmr frequency indicating that backbone motions are fast compared to the nmr frequencies. However, as the temperature is increased above 250 K, relaxation is significantly more efficient at the lowest frequency, which shows, in addition, the presence of motions that are slow compared to the nmr frequencies. Using the values of S2 determined from the residual quadrupole coupling and a model-free relaxation formalism that allows for fast and slow internal motions, we conclude that these slow motions have correlation times in the range of 0.1 to 1.0 microsecond and are effectively frozen out at 250 K where fast motions of the amide planes with approximately 15 ps effective correlation times and 9 degrees rms amplitudes dominate relaxation. The fast internal motions increase slightly in amplitude as the temperature rises toward 290 K, but the correlation time, as is also observed in solution nmr studies of RNase H, is approximately constant. These findings are consistent with hypotheses of dynamic glass transitions in hydrated proteins arising from temperature-dependent damping of harmonic modes of motion above the transition point.

17O NMR and crystalline hydrates.

17O NMR is complementary to 2H NMR for studies of hydrate structure and dynamics in solids, insofar as the quadrupole coupling parameters can be accurately determined. At a Larmor frequency of 34.2 MHz, the effects of a large 17O quadrupole coupling (e2qQ/h approximately 7 MHz) on both the orientational dependence of the central transition resonance frequency and its observation by cross-polarization are examined in powders and a single crystal of oxalic acid dihydrate. Comparison with the exact energies shows that the variation of the central transition frequency with tensor orientation is accurately described as a second-order perturbation, even when e2qQ/h and the Larmor frequency are comparable. The combined chemical and quadrupole-induced shifts of the central transition frequency are separated by their distinct transformation properties, and we report the H2(17)O quadrupole coupling tensor in alpha-oxalic acid dihydrate, which is compared with the hydrogen bonding structure. Excitation of the central transition by cross-polarization is examined in terms of the 17O nutation frequency. Distortions in powder patterns and the loss of lines in single-crystal spectra owing to the orientational dependence of the first-order quadrupole coupling are verified, and simple methods for mitigating the problem in single-crystal and powder experiments are presented.

Deuterium quadrupole coupling in N-acetylglycine and librational dynamics in solid poly(gamma-benzyl-L-glutamate).

To study the dynamics of peptide groups in solid proteins, we have accurately determined the principal components and molecular orientation of the electric field gradient tensor for the exchangeable deuterons in monoclinic N-acetylglycine by single-crystal deuterium nuclear magnetic resonance. These results are compared with the principal components of the amide deuterons in solid poly(gamma-benzyl-L-glutamate) measured in powder samples over a wide temperature range (140-400 K). The comparison indicates that in the solid polypeptide the N-D bonds undergo a small-amplitude torsional reorientation (libration) perpendicular to the peptide plane. To estimate dynamic rates, longitudinal relaxation times (T1 values) are reported for N-acetylglycine and poly(gamma-benzyl-L-glutamate). T1 values for the carboxyl and amide deuterons in N-acetylglycine are approximately 100 s, whereas for the amide deuterons in the polypeptide T1 approximately 1 s, also indicating that the N-D bonds are not stationary in the polypeptide. We determine from the reduced quadrupole coupling tensor the mean-square amplitude for the libration and show that it increases linearly with temperature. A simple qualitative theory for the relaxation times is presented on the basis of the assumption that the N-D reorientation is described either as a diffusion process in a square well or as a damped Langevin oscillator with a harmonic restoring force. The conclusion is that the short relaxation times of the polypeptide amide deuterons result from substantial frictional effects on reorientation that increase with temperature.

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)

Molecular dynamics and conformation in the gel and liquid-crystalline phases of phosphatidylethanolamine bilayers.

Solid-state deuterium and carbon-13 nuclear magnetic resonance (NMR) spectra have been used to study the molecular dynamics and conformation of dipalmitoylphosphatidylethanolamine (DPPE) in both the gel (L beta) and liquid-crystalline (L alpha) phases. For this purpose DPPE was labeled with 13C in the carbonyl group of the sn-2 chain and with 2H at three different positions--4, 8, and 12--of the sn-2 chain, at the 2 position of the glycerol backbone, and at the 1 position of the ethanolamine head group. The 13C carbonyl and 2H chain spectra indicate that in the gel phase the DPPE molecules are diffusing about their long axes at rates of 10(5)-10(6) s-1 and the acyl chains are in an approximately all-trans conformation. The glycerol backbone spectra suggest that the backbone is in a gauche conformation in the gel state, rather than a trans conformation such as found in single crystals. The head group spectra in the gel phase are broad, featureless lines of about 20-kHz width. At the L beta leads to L alpha phase transition several changes take place. As is well-known, the chains disorder, and fast long-axis rotational diffusion begins, which results in the sharp, axially symmetric L alpha phase 2H spectra, which are a factor of 2 narrower than those observed in the L beta phase. The head group spectra also sharpen substantially at the transition, although their total width remains approximately constant. The invariance of the spectral width suggests that the average head group conformation is similar in both phases. However, the sharper spectra seen in the L alpha phase indicate that the rates of the head group motions in this phase are at least 3 orders of magnitude faster than those in the L beta phase. Thirdly, the 2H spectra of the glycerol backbone labeled DPPE narrow by a factor of about 4, and we believe this is due to a conformational change in this region of the molecule. Consistent with this interpretation is the fact that the powder pattern exhibited by the sn-2 13C = O in the L beta phase collapses to an isotropic-like line at the phase transition.

Phase equilibria, molecular conformation, and dynamics in phosphatidylcholine/phosphatidylethanolamine bilayers.

Solid-state 13C and 2H NMR experiments have been used to examine the phase equilibria and dynamic structure of binary mixtures of dipalmitoylphosphatidylcholine (DPPC) and dipalmitoylphosphatidylethanolamine (DPPE). The experiments rely on changes in the 13C and 2H spectra of sn-2 13C = O labeled and 2H chain and head group labeled lipids at phase boundaries. In particular, broad powder patterns are observed in the gel state, and these patterns narrow dramatically in the liquid-crystalline phase. In the two-phase region a superposition of the gel-phase and liquid-crystalline-phase spectra is observed, which results from the presence of large domains in the DPPC/DPPE system. The appearance of the liquid-crystalline component and the disappearance of the gel component permit the solidus and liquidus curves of the phase diagram to be located accurately. Furthermore, a comparison of the temperature dependence of the 13C and 2H spectra shows the phase transition mechanism to be a function of composition. This result, together with other evidence, supports the hypothesis that the gel-state lattice changes from the L beta or P beta to the L beta configuration with increasing DPPE content. Finally, a detailed examination of the composition dependence of the spectra provides evidence for nonideal mixing.

Solid-state carbon-13 nuclear magnetic resonance of the lecithin gel to liquid-crystalline phase transition.

The temperature dependence of the 13C NMR spectra of dipalmitoylphosphatidylcholine (DPPC) which has been 13C labeled at the carbonyl position of the sn-2 chain, 2-[1-13C]DPPC, is reported. In the L beta' phase an axially symmetric spectrum of 112-ppm breadth is observed, and this transforms to an isotropic-like line ((delta sigma) approximately 7 ppm) in the L alpha phase. In the intermediate P beta phase a temperature-dependent superposition of these spectra is observed, which suggests that this phase exhibits microscopic structural and dynamical properties of both the L beta' and L alpha phases. An analysis of the spectral line shapes leads to the conclusion that the appearance of the isotropic-like line in the P beta' phase is primarily due to a conformational change at the sn-2 carbonyl which is complete at the main transition. Increased rates of axial diffusion in the P beta' phase may contribute to the narrowing.

Aliphatic groups of sperm whale myoglobin: 13C NMR study.

The aliphatic region of the 13C NMR spectrum of sperm whale cyanoferrimyoglobin has been examined at 67.9 MHz. Fifty partially resolved or well-resolved resonances, representing at least half of the aliphatic carbons in the molecule, are observed in the spectral region from 9 to 29 ppm downfield of tetramethylsilane. Analyses of the spin lattice relaxation times (T1) and nuclear Overhauser enhancements for these resonances reveal considerable motion freedom of the aliphatic side chains. In the spectral region from 9 to 15 ppm, eight single carbon resonances are observed and tentatively assigned to Cdelta 1 of eight of the nine isoleucine residues. In at least five cases the reorientational motion of the isoleucine side chains could not be characterized solely by rotation of the Cdelta 1 methyl groups. The simplest model consistent with the data is a restricted diffusion model with two degrees of internal rotation [Wittenbort, R. J. & Szabo, A. (1978) J. Chem. Phys. 69, 1722--1736]. In light of the packing densities within the myoglobin molecule these results are taken to imply concerted motions of the buried aliphatic residues.

The quantitation of carbamino adduct formation of angiotensin II and bradykinin.

The two equilibrium constants that define the extent of carbamino adduct formation with amines for all values of pH and PCO2 are determined for the alpha-amino groups of the peptide hormones angiotensin II(AII) and bradykinin (BK) by nuclear magnetic resonance techniques. From these constants the variation of carbamino adduct formation has been calculated over the pH range 6.60--8.00 with variable PCO2, and the results are superimposed upon standard pH-bicarbonate diagrams. PCO2, and the results are superimposed upon standard pH-bicarbonate diagrams. The mole fraction, Z, of carbamino adduct form of AII or BK shows a maximum variation in going from metabolic alkalosis, Z congruent to 0.30, to metabolic acidosis, Z congruent to 0.02, with Z near 0.2 for normal acid-base conditions. Adduct formation to hormone may alter the biological effect of the hormone (a) by limiting proteolysis, particularly at the amino-terminal, (b) by altering hormone binding affinity to specific receptors, or (c) by converting the hormone to an antagonist which binds to receptor but does not activate subsequent metabolic events. The requirements for any of these mechanisms to operate are examined in terms of simple equilibrium considerations, and experimental evidence of inhibition of an aminopeptidase model system is presented. These results are consistent with the hypothesis that regulation of some physiological processes through formation of carbamino adduct of peptide hormones is possible.

Quantitative determination of carbamino adducts of alpha and beta chains in human adult hemoglobin in presence and absence of carbon monoxide and 2,3-diphosphoglycerate.

The principal component of normal adult human hemoglobin was equilibrated under various conditions with 13CO2. Quantitative analysis of the carbamino resonance intensities over the pH range of 6.5 to 9.0 shows that the effects of conversion from the deoxy to the liganded state in reducing the carbamino adduct formation occur predominantly at Val-1beta. Analysis of the pH dependence of carbamino formation at constant total carbonates yields values of pKz and pKc for Val-1beta and Val-1alpha in the deoxy and liganded conditions. In contrast to the Val-1beta as the allosteric site for CO2, the Val-1alpha site is shown to be primarily an alkaline Bohr group. 2,3-Diphosphoglycerate is shown to reduce substantially the Val-1beta carbamino resonance intensity in deoxyhemoglobin. Evidence for 2,3-diphosphoglycerate effects in carbon monoxide hemoglobin at both Val-1alpha and Val-1beta sites is presented. Enhanced carbamino formation in carbon monoxide hemoglobin at Val-1beta is observed at pH values less than 7.8. Finally, chemical exchange analysis of the spectra shows the release rate of the deoxy Val-1alpha carbamino adduct to be greater than that for deoxy Val-1beta. At pH 7.47 k-1obs,beta congruent to 1.0 and k-1obs, alpha congruent to 11.0 s-1.

Carbon 13 resonances of 13CO2 carbamino adducts of alpha and beta chains in human adult hemoglobin.

The principal component of normal adult human hemoglobin Ao, was equilibrated under various conditions with 13CO2. In addition, derivatives containing specifically carbamylated NH2-terinal groups in alpha or beta chains, or both, were prepared by treatment with cyanate, and equilibrated likewise to allow the identification of specific resonances observed by 13C nuclear magnetic resonance. In deoxyhemoglobin, a resonanance at 29.2 ppm upfield of external CS2 was assigned to the alpha chain terminal adduct, and one at 29.8 ppm to the beta chain terminal adduct. In the liganded state as the CO derivative, the terminal adduct on both chains showed a common resonance position at 29.8 ppm. Small effects of pH on the resonance positions were observed. Under certain conditions, a resonance was observed at 33.4 ppm, probably not ascribable to a carbamino compound. A carbamino resonance that became prominent at higher pH was found at 28.4 ppm, and is tentatively ascribed to one or more adducts on epsilon amino groups. The beta chain resonances in particular are minimized by the presence of inositol hexaphosphate or 2,3-diphosphoglycerate. Quantitative analysis of the resonance intensities shows that the effects of conversion from the deoxy to the liganded state in reducing the degree of carbamino adduct is much more pronounced for the beta than for the alpha chains.