Anisotropic 2H-nuclear magnetic resonance spin-lattice relaxation in cerebroside- and phospholipid-cholesterol bilayer membranes.

The axially symmetric powder pattern 2H-nuclear magnetic resonance (NMR) lineshapes observed in the liquid crystalline phase of pure lipid or lipid/cholesterol bilayers are essentially invariant to temperature, or, equivalently, to variations in the correlation times characterizing C-2H bond reorientations. In either of these melted phases, where correlation times for C-2H bond motions are shorter than 10(-7) s, information on the molecular dynamics of the saturated hydrocarbon chain would be difficult to obtain using lineshape analyses alone, and one must resort to other methods, such as the measurement of 2H spin-lattice relaxation rates, in order to obtain dynamic information. In pure lipid bilayers, the full power of the spin-lattice relaxation technique has yet to be realized, since an important piece of information, namely the orientation dependence of the 2H spin-lattice relaxation rates is usually lost due to orientational averaging of T1 by rapid lateral diffusion. Under more favorable circumstances, such as those encountered in the lipid/cholesterol mixtures of this study, the effects of orientational averaging by lateral diffusion are nullified, due to either a marked reduction (by at least an order of magnitude) in the diffusion rate, or a marked increase in the radii of curvature of the liposomes. In either case, the angular dependence of 2H spin-lattice relaxation is accessible to experimental study, and can be used to test models of molecular dynamics in these systems. Simulations of the partially recovered lineshapes indicate that the observed T1 anisotropies are consistent with large amplitude molecular reorientation of the C-2H bond among a finite number of sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Accessibility and dynamics of nitroxide side chains in T4 lysozyme measured by saturation recovery EPR

Long pulse saturation recovery electron paramagnetic resonance spectroscopy is applied to the investigation of spin-labeled side chains placed along a regular helix extending from 128 to 135 in T4 lysozyme. Under an argon atmosphere, analysis of the exponential saturation recovery curves gives the spin-lattice relaxation rates of the nitroxides, which depend on the nitroxide side-chain dynamics. In the presence of the fast-relaxing paramagnetic reagents O(2) or NiEDDA, global analysis of the saturation recovery provides the spin-lattice relaxation rates as well as the Heisenberg exchange rates of the nitroxide with the reagents. As previously shown with power saturation methods, such exchange rates are direct measures of the solvent accessibility of the nitroxide side chains in the protein structure. The periodic dependence of the spin-lattice relaxation rates and the exchange rates along the 128-135 sequence reveal the presence of the helical structure, demonstrating the use of these parameters in structure determination. In general, multiple exponentials are required to fit the saturation recovery data, thus identifying multiple states of the side chain. In one case, multiple conformations detected in the spectrum are not evident in the saturation recovery, suggesting rapid exchange on the timescale of spin-lattice relaxation.     

Temperature dependence of the electronic spin-lattice relaxation time in a 2-iron-2-sulfur protein.

The ferredoxins are characterized by a strong temperature dependence of the electronic spin-lattice relaxation time T1. The measurement of this dependence above the liquid nitrogen temperature has been presented in earlier work [1] for the 2-iron-2-sulfur ferredoxin of the blue green alga Spirulina maxima. The different relaxation mechanisms which could be efficient in this range were briefly discussed. In the present paper, we extend the measurement of the temperature dependence of T1 to the low temperature range 1.25 to 30 K. From 1.25 K to 13 K, T1 is obtained by the saturating pulse method, whereas the continuous saturation method is used from 8 K to 30 K. The experimental conditions concerning these methods are discussed. The analysis of the temperature dependence curve over the whole range 1.25 K to 133 K shows clearly that different regions must be distinguished. For each region the possible relaxation processes and the corresponding vibrational modes are discussed.     

Assignment of the 13C-nmr spectrum of Hippurin, a novel furospirostane: observation of both ends of the axis of anisotropic reorientation through the measurement of spin-lattice relaxation times

Anisotropic reorientation of 3 alpha-substituted steroids has been well documented. Assignment of the 13C-nmr spectrum of Hippurin, a novel furospirostane and the measurement of the spin-lattice (T1) relaxation times has established both points in the molecule through which the axis of anisotropic reorientation passes, specifically the 3- and 24-positions.     

Proton NMR T1, T2, and T1 rho relaxation studies of native and reconstituted sarcoplasmic reticulum and phospholipid vesicles.

The phospholipids protons of native and reconstituted sarcoplasmic reticulum (SR) membrane vesicles yield well-resolved nuclear magnetic resonance (NMR) spectra. Resonance area measurements, guided by the line shape theory of Bloom and co-workers, imply that we are observing a large fraction of the lipid intensity and that the protein does not appear to reduce the percent of the signal that is well resolved. We have measured the spin-lattice (T1) and spin-spin (T2) relaxation rates of the choline, methylene, and terminal methyl protons at 360 MHz and the spin-lattice relaxation rate in the rotating frame (T1 rho) at 100 MHz. Both the T1 and T2 relaxation rates are single exponential processes for all of the resonances if the residual water proton signal is thoroughly eliminated by selective saturation. The T1 and T2 relaxation rates increase as the protein concentration increases, and T2 rate decrease with increasing temperature. This implies that the protein is reducing both high frequency (e.g., trans-gauche methylene isomerizations) and low frequency (e.g., large amplitude, chain wagging) lipid motions, from the center of the bilayer to the surface. It is possible that spin diffusion contributes to the effect of protein on lipid T1's although some of the protein-induced T1 change is due to motional effects. The T2 relaxation times are observed to be near 1 ms for the membranes with highest protein concentration and approximately 10 ms for the lipids devoid of protein. This result, combined with the observation that the T2 rates are monophasic, suggests that at least two lipid environments exist in the presence of protein, and that the lipids are exchanging between these environments at a rate greater than 1/T2 or 10(3) s-1. The choline resonance yields single exponential T1 rho relaxation in the presence and absence of protein, whereas the other resonances measured exhibit biexponential relaxation. Protein significantly increases the single T1 rho relaxation rate of the choline peak while primarily increasing the T1 rho relaxation rate of the more slowly relaxing component of the methylene and methyl resonances.     

Influence of methanol on the rotational diffusion of poly(L-lysine) in the helix–coil transition

¹³C spin-lattice relaxation times of poly(L -lysine) have been obtained at 67.9 MHz in aqueous solution and in a mixed solvent (40% methanol/60% water). A concomitant determination of the conformation by CD permits the correlation of conformation and rotational diffusion of the polymer. The dependence on pH of the spin-lattice relaxation times of the ¹³C^α and the side-chain carbon resonances reflects the diffusional motion in the random-coil conformation, in the helix–coil transition, and in the conformation of the α-helix. In the mixed solvent the reorientational correlation time of the C^α-H^α vector increases from τ = 0.37 nsec (random coil) to τ = 12.0 nsec (α-helix). In aqueous solution the correlation time of this vector increases from τ = 0.33 nsec (random coil) to τ ? 11 nsec. The reorientation rates of the side-chain methylene groups in the two solvents are markedly different. The reorientation of all methylene groups is reduced in the mixed solvent.     

Proton NMR spin grouping and exchange in dentin.

The nuclear magnetic resonance spin-grouping technique has been applied to dentin from human donors of different ages. The apparent T2, T1, and T1 rho have been determined for natural dentin, for dentin which has been dried in vacuum, and for dried dentin which has been rehydrated in an atmosphere with 75% relative humidity. All apparent spin relaxation has been analyzed for exchange between the spin groups in which the dentin protons exist; the analyses incorporate the results of selective inversion recovery T1 measurements which better probe the effects of exchange. The exchange analyses of the high fields and rotating frame spin-lattice relaxation have also been correlated to determine uniquely the inherent relaxation parameters of the proton spin groups constituting the dentin magnetization. The natural dentin contains protons on water, protein, and hydroxy apatite; these spins contribute 50%, 45%, and 5% to the total dentin proton magnetization, respectively. The water exists in three distinct environments, the dynamics of each environment has been modeled. In the natural dentin 30% of the water undergoes uni-axial reorientation. 52% of the water has similar relaxation characteristics to bound water hydrating a large molecule, and the majority of the remaining water acts as bulk water undergoing isotropic reorientation. The results are independent of the age of the donor.     

Magnetic cross-relaxation among protons in protein solutions.

The magnetic spin-lattice relaxation rates of solvent water nuclei are known to increase upon addition of diamagnetic solute protein. This enhancement of the relaxation rate is a function of magnetic field, and the orientational relaxation time of the protein molecules can be deduced from analysis of the field-dependent relaxation rates. Although the nature of the interactions that convey information about the dynamics of protein motion to the solvent molecules is not established, it is known that there is a contribution to the relaxation rates of solvent protons that plays no role in the relaxation of solvent deuterons and 17O nuclei. We show here that the additional interaction arises from a cross-relaxation process between solvent and solute protons. We introduce a heuristic three-parameter model in which protein protons and solvent protons are considered as two separate thermodynamic systems that interact across the protein-solvent interface. The three parameters are the intrinsic relaxation rates of each system and a cross-relaxation term. The sign of the latter term must always be positive, for all values of magnetic field, in order for magnetization energy to flow from the hotter to the cooler system. We find that the magnetic field-dependence of the cross-relaxation contribution is much like that of the remaining solvent proton relaxation, i.e., about the same as the deuteron relaxation field dependence. This finding is not compatible with the predictions of expressions for the cross-relaxation that have been used by other authors, but not applied to data over a wide range of magnetic field strength. The model predicts that the relaxation behavior of both the protein protons and the solvent protons is the sum of two exponentials, the relative contributions of which would vary with protein concentration and solvent isotopic composition in a fashion suggestive of the presence of two classes of protein protons, when there is in reality only one. This finding has immediate implications for the interpretation of published proton relaxation rates in complex systems such as tissues; these data should be reexamined with cross-relaxation taken into account.     

Conformational flexibility of angiotensin II. A carbon-13 spin-lattice relaxation study.

Carbon-13 spin-lattice relaxation times (T1) have been determined for the carbon in the octapeptide hormone [5-isoleucine]-angiotensin II in aqueous solution. Two possible models for molecular motion are considered: isotropic overall motion of the hormone with internal motion of some residues and anisotropic overall molecular motion. The data are interpreted in detail using the former model. The alpha carbons of the peptide backbone are all equally restricted in their motion. The correlation time for overall molecular reorientation, calculated from an everage T1 value of 95 msec for the alpha carbons in the peptide backbone, is ca. 5 times 10-10 sec. The carbons in the side chains are more mobile than those in the peptide backbone, with the exception of the side chain of the Tyr residue which does not undergo rapid segmental motion. We propose that [5-isoleucine]-angiotensin II has a restricted backbone conformation and that the alpha carbons of the N- and C-terminal residues are constrained to nearly the same extent as the remaining alpha carbons in the peptide backbone. Chemical shift data indicate that the Pro residue adopts the trans conformation about the His-Pro bond and that the imidazole ring of His has a strong preference for the N-tau -H tautomer.     

Conformational flexibility of luteinizing hormone-releasing hormone in aqueous solution. A carbon-13 spin-lattice relaxation time study.

The carbon-13 nuclear magnetic resonance (13C NMR) spectra of luteinizing hormone-releasing hormone (LH-RH) and lower homologous peptides have been assigned in aqueous solutions at various pH values. 13C spin-lattice relaxation times (T1) have been measured for all proton-bearing carbons at 25.2 and 67.9 MHz. From the T1 data the rates of overall molecular motion and intramolecular motion of side chains have been estimated. LH-RH is a flexible molecule in solution, having segmental motion along the backbone as well as in the nonaromatic side chains.     

31P NMR relaxation studies of the activation of the coenzyme phosphate of glycogen phosphorylase. The role of motion of the bound phosphate.

Spin-lattice and spin-spin relaxation rates (1/T1 and 1/T2) have been determined for the catalytically essential coenzyme phosphate at the active site of glycogen phosphorylase in both activated (R state) and inactive (T state) conformations of the enzyme. Dipolar contributions to 31P relaxation due to exchangeable protons on the phosphate group have been determined by measurement of relaxation rates at different concentrations of H2O and D2O, and field dependence studies have been performed to estimate the contribution of chemical shift anisotropy to the remaining 31P relaxation in D2O. At 109 MHz, dipolar relaxation from exchangeable protons was found to account for 50% of the spin-lattice relaxation for activated phosphorylase in 75% H2O, the remainder being due to chemical shift anisotropy. The spin-lattice relaxation rates in D2O for R-state glycogen phosphorylase are very similar to those measured for other proteins of very different size such as actin (Brauer, M., and B. D. Sykes, 1981, Biochemistry. 20:6767-6775), alkaline phosphatase (Coleman, J. E., I. D. Armitage, J. F. Chlebowski, J. D. Otvos, and A. J. M. S. Uiterkamp, 1979), and phosphoglucomutase (Rhyu, G. I., W. J. Ray, Jr., and J. L. Markley, 1984, Biochemistry. 23:252-260). In inactive (T state) phosphorylase the spin-lattice relaxation rates were almost an order of magnitude slower, while the spin-spin relaxation rates were essentially identical. These results have been analyzed by calculating the theoretically expected 31P relaxation rates in the presence of internal motions that are included in the relaxation calculation using the model-free approach of Lipari and Szabo (1982, J. Am. Chem. Soc. 104:4564-4559).(ABSTRACT TRUNCATED AT 250 WORDS)     

Dipolar NMR relaxation of nonprotonated aromatic carbons in proteins. Structural and dynamical effects.

The crystal structure and a 96-ps molecular dynamics simulation used to analyze structural and motional contributions to spin-lattice (T1) relaxation times of phenylalanine and tyrosine C gamma carbons of the pancreatic trypsin inhibitor. The H beta and H delta protons geminal to C gamma are calculated to account for approximately 80% of the dipolar relaxation for each residue. Experimental T1 values for the phenylalanine residues obtained at 25 MHz are observed to be 15-25% longer than estimates based on the rigid crystal structure. It is shown how an increase in T1 can be related to order parameters for the picosecond motional averaging of the important C,H dipolar interactions, and how these order parameters can be calculated from a protein molecular dynamics trajectory.     

ATP synthesis and degradation rates in the perfused rat heart. 31P-nuclear magnetic resonance double saturation transfer measurements.

A limitation of magnetization transfer techniques for studying enzyme kinetics in vivo has been the difficulty of treating systems with more than two exchanging species. This problem was addressed in the original papers describing saturation transfer. Since then, a number of approaches have been devised to study these complex situations. Here, we present a method based on the transient saturation transfer experiment in which spin-lattice relaxation time constants and reaction rates are obtained from the same magnetization transfer data. This technique is particularly suitable for biological samples. We apply the method to evaluate flux balance in the three-site linear exchange network composed of ATP, creatine phosphate, and inorganic phosphate in the isolated, perfused rat heart and show that the method yields reasonable values for the reaction velocities of ATP synthesis and degradation.     

The influence of glycyl residues on the flexibility of peptide hormones in solution. A 13C-nuclear-magnetic-resonance study of luteinizing hormone-releasing hormone (luliberin) and its des-glycinamide10 N-ethylamide analog.

13C nuclear magnetic resonance spectroscopy in used to gain information on the flexibility of the backbone in peptide hormones and peptide hormone analogs. 13C spin-lattice relaxation times (T1) were measured on luliberin, the luteinizing-hormone-releasing hormone and des(Gly-NH2)10-luliberin-N-ethylamide in aqueous solution at 25.2 and 67.9 MHz at temperatures of 32 degrees, 40 degrees and 55 degrees C. The 13C spin-lattice relaxation times indicate increased flexibility of the peptide backbone in the immediate environment of glycyl residues in luliberin (less than Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2) and the hormone analog des(Gly-NH2)10-luliberin-N-ethylamide (less than Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-NH-CH2CH3) in aqueous solutions. 13 C NMR spectroscopy is shown to be a sensitive technique for monitoring the time-averaged conformational flexibility of peptides in solution. Activation energies (Ea) of about 25 kJ/mol were obtained for rotational reorientation of non-terminal alpha-carbons in the peptide backbone. Rotation of methyl groups was characterized by an Ea of 9.6 kJ/mol whereas reorientation of the N-terminal pyroglutamyl residue showed an Ea value of 14.6 kJ/mol. The Ea values of individual carbons in the side-chains of prolyl, arginyl and leucyl residues in the peptides were similar to those obtained for the alpha-carbon of the same amino acid residue in the peptide backbone of the hormones.     

Molecular motions and phase transitions. NMR relaxation times studies of several lecithins.

The spin-lattice relaxation time, T1, and the dipolar energy relaxation time, TD, were measured as a function of temperature. The materials studied were samples of anhydrous L-dipalmitoyl lecithin, DL-dipalmitoyl lecithin, L-dimyristoyl lecithin, DL-dimyristoyl lecithin and their monohydrates, and of anhydrous egg yolk lecithin. It is shown that TD is a much more sensitive parameter than T1 for the determination of the Chapman phase transition. Comparison between T1 and TD provides information about new types of slow molecular motions below and above the phase transition temperature. It is suggested that the relaxation mechanisms for T1 and TD in the gel phase are governed by segmental motion in the phospholipid molecule. A new metastable phase was detected in dimyristoyl lecithin monohydrates. This phase could only be detected from the dipolar energy relaxation times.     

Dynamics of the phosphate group in phospholipid bilayers. A 31P nuclear relaxation time study.

The spin-lattice relaxation time of the 31P nucleus in the phosphate group of egg yolk phosphatidylcholine multilamellar dispersions has been investigated at four resonant frequencies (38.9, 81.0, 108.9, and 145.7 MHz) in the temperature range from -30 degrees to 60 degrees C. The observed frequency dependence of the relaxation indicates that both dipolar relaxation and relaxation due to anisotropic chemical shielding are significant mechanisms. The experimental data have thus been modeled assuming both mechanisms and the analysis has allowed the contribution of each to the relaxation to be determined along with the correlation time for the molecular reorientation as a function of temperature. Dipolar relaxation was found to dominate at low nuclear magnetic resonance frequencies while at high frequencies the anisotropic chemical shift dominates. The correlation time of the phosphate group is on the order of 10(-9) s at 60 degrees C and increases to approximately 10(-7) s at -30 degrees C. It is observed that the freezing of the buffer which occurs at approximately -8 degrees C has a significant effect on the phosphate group reorientation. This effect of the freezing is to change the activation energy for the phosphate group reorientation from 16.9 KJ/mol above -8 degrees C to 32.5 KJ/mol below -8 degrees C.     

NMR investigations of Mn(II)-leucine-enkephalin complexes in [2H6]-DMSO solution.

Structural and kinetic features of the Mn(II)-Leu-enkephalin binding equilibria were delineated by measuring 13C and 1H NMR spin-lattice relaxation rates. The temperature dependence of such rates showed that some carbons were experiencing slow exchange regimes such that kinetic parameters at room temperature could be calculated (k(off) = 1400 sec-1, delta H* = 12.0 kcal/mol, delta S* = -9.9 e.u.). The paramagnetic rates of fast exchanging carbons were interpreted by the Solomon-Bloembergen-Morgan theory to provide structural parameters. The terminal carboxyl and amino groups were shown to be the binding sites. The motional correlation time (tau c = 0.6 nsec at 298 K) was calculated by measuring selective and double-selective 1H spin-lattice relaxation rates for the free peptide. The number of coordinated ligands was evaluated by considering the distance of the Leu CO in the complex at 2.54 A, as shown by molecular models. Finally, carbon-Mn(II) distances were calculated and the molecular model of the 1:1 complex was built.     

Spin-lattice relaxation of water protons in plant and animal cells

NMR-spin echo method has been used to study spin-lattice relaxation time of protons T1 in plant and animal cells ?? muscle tissue of fish, the cells of which unlike plant cells have no developed system of vacuoles, plastids and a solid cell wall. According to the values of T1 time a new NMR parameter K, a coefficient of relaxation effectiveness of a cell structure, has been calculated. This parameter can be used for quantitative characterization of the influence of different cell structures, the tissue water interact with, for a time of spin-lattice relaxation of water protons. It has been ascertained that the values of K coefficient in animal tissue and in storing tissues of some plants differ little; it may be stipulated by permanent transmembrane water exchange which occurs at high rate in the living cell. It has been concluded that there exists a certain similarity between water state in protoplast of plant and animal cells.     

Internal motions in B- and Z-form poly(dG-dC).poly(dG-dC): 1H NMR relaxation studies

Proton NMR relaxation measurements are used to compare the molecular dynamics of 60 base pair duplexes of B- and Z-form poly(dG-dC).poly(dG-dC). The relaxation rates of the exchangeable guanine imino protons (Gim) in H2O and in 90% D2O show that below 20 degrees C spin-lattice relaxation is exclusively from proton-proton magnetic dipolar interactions while proton-nitrogen interactions contribute about 30% to the spin-spin relaxation. The observation that the spin-lattice relaxation is nonexponential and that the initial spin-lattice relaxation rate of the Gim, G-H8 and C-H6 protons depends on the selectivity of the exciting pulse shows that spin-diffusion dominates the spin-lattice relaxation. The relaxation rates of the Gim, C-H5, and C-H6 in B- and Z-form poly(dG-dC).poly(dG-dC) cannot be explained by assuming the DNA behaves as a rigid rod. The data can be fit by assuming large-amplitude out of plane motions (+/- 30-40 degrees, tau = 1-100 ns) and fast, large-amplitude local torsional motions (+/- 25-90 degrees, tau = 0.1-1.5 ns) in addition to collective torsional motions. The results for the B and Z forms show that the rapid internal motions are similar and large in both conformations although backbone motions are slightly slower, or of lower amplitude, in Z DNA. At high temperatures (greater than 60 degrees C), imino proton exchange with solvent dominates the spin-lattice relaxation of B-form poly(dG-dC).poly(dG-dC), but in the Z form no exchange contribution (less than 2 s-1) is observed at temperatures as high as 85 degrees C. Conformational fluctuations that expose the imino protons to the solvent are strikingly different in the B and Z forms. The results obtained here are compared with those previously reported for poly(dA-dT).poly(dA-dT).     

Carbon-13 NMR relaxation studies of pre-melt structural dynamics in [4-13C-uracil] labeled E. coli transfer RNAIVal.

We report 67.8 MHz carbon-13 spin-lattice relaxation studies on [4-13C-uracil] labeled tRNAIVal purified from E. coli SO-187. Following 13C-enriched C4 carbonyl resonances from modified and unsubstituted uridines scattered throughout the polymer backbone enables us to determine dynamical features in both loop and helical stem regions. The experimental results have been analyzed in terms of a model of isotropic overall molecular reorientation. "Anomalous" residues for which the experimental data cannot be accounted for in terms of the model provide an assessment of local and regional properties. Thus, "native" tRNAIVal under physiological conditions of magnesium (10 mM) and temperature (20 degrees - 40 degrees C), exhibits the following characteristics: 1) uridines held rigidly in helical stems and tertiary interactions display correlation times for rotational reorientation of 15-20 nsecs, typical for overall tRNA motion; 2) uridines in loops such as the wobble residue uridine-5-oxyacetic acid (V34) are quite accessible to solvent; moreover V34 and another loop residue, D17, exhibit local mobility; 3) the tertiary interactions involving 4-thio uridine (s4U8) and A14 and ribothymidine (rT54) and A58 are weakened as temperature increases.