Dipole-dipole Relaxation Research Articles

Carbon-13 NMR Longitudinal Relaxation Time Study of an Ionophoric 1,3-Alternate-Shaped Calix[4]arene Ester

The carbon-13 NMR longitudinal relaxation times (T1) studies on the 1,3-alternate-shaped calix[4]arene possessing two ester groups 1 in the absence and the presence of Na+ were carried out for the first time. The T1 values were measured under proton-noise-decoupling conditions by the inversion-recovery technique. The T1-value of 1 is primarily governed by dipole-dipole relaxation. When calixarene 1 forms an ensemble with Na+, the T1-values for all carbons are decreased. This suggests that the molecular motion of species 1 is efficiently frozen through complexation with Na+. Eventually, Na+ ion plays an important role as a template ion specifically to freeze the fluctuational motion of two ethoxycarbonylmethoxybenzene units of 1.

Structure and Dynamics of a Dihydrogen/Hydride Ansa Molybdenocene Complex

In contrast to [Cp(2)MoH(3)](+), which is a thermally stable trihydride complex, the ansa-bridged analogue [(eta-C(5)H(4))(2)CMe(2)MoH(H(2))](+) (1) is a thermally labile dihydrogen/hydride complex. Partial deuteration of the hydride ligands allows observation of J(H)(-)(D) = 11.9 Hz in 1-d(1) and 9.9 Hz in 1-d(2) (245 K), indicative of a dihydrogen/hydride structure. There is a slight preference for deuterium to concentrate in the dihydrogen ligand. A rapid dynamic process interchanges the hydride and dihydrogen moieties in complex 1. Low temperature (1)H NMR spectra of 1 give a single hydride resonance, which broadens at very low temperature due to rapid dipole-dipole relaxation (T(1) = 23 ms (750 MHz, 175 K) for the hydride resonance in 1). Low temperature (1)H NMR spectra of 1-d(2) allow the observation of decoalescence at 180 K into two resonances. The bound dihydrogen ligand exhibits hindered rotation with DeltaG(150) = 7.4 kcal/mol, but H atom exchange is still rapid at all accessible temperatures (down to 130 K). Density functional calculations confirm the dihydrogen/hydride structure as the ground state for the molecule and give estimates for the energy of two hydrogen exchange processes in good agreement with experiment. The presence of the C ansa bridge is shown to decrease the ability of the metallocene fragment to donate to the hydrogens, thus stabilizing the (eta(2)-H(2)) unit and modulating the barrier to H(2) rotation.

Beyond the T1 limit: singlet nuclear spin states in low magnetic fields.

Low-field nuclear spin singlet states may be used to store nuclear spin order in a room temperature liquid for a time much longer than the spin-lattice relaxation time constant T1. The low-field nuclear spin singlets are unaffected by intramolecular dipole-dipole relaxation, which is generally the predominant relaxation mechanism. We demonstrate storage of nuclear spin order for more than 10 times longer than the measured value of T1. This phenomenon may facilitate the development of nuclear spin hyperpolarization methods and may allow the study of motional processes which occur too slowly for existing NMR techniques. This is the first time that the memory of nuclear spins has been extended well beyond the T1 limit in a system lacking intrinsic magnetic equivalence.

Fast structure-based assignment of 15N HSQC spectra of selectively 15N-labeled paramagnetic proteins.

A novel strategy for fast NMR resonance assignment of (15)N HSQC spectra of proteins is presented. It requires the structure coordinates of the protein, a paramagnetic center, and one or more residue-selectively (15)N-labeled samples. Comparison of sensitive undecoupled (15)N HSQC spectra recorded of paramagnetic and diamagnetic samples yields data for every cross-peak on pseudocontact shift, paramagnetic relaxation enhancement, cross-correlation between Curie-spin and dipole-dipole relaxation, and residual dipolar coupling. Comparison of these four different paramagnetic quantities with predictions from the three-dimensional structure simultaneously yields the resonance assignment and the anisotropy of the susceptibility tensor of the paramagnetic center. The method is demonstrated with the 30 kDa complex between the N-terminal domain of the epsilon subunit and the theta subunit of Escherichia coli DNA polymerase III. The program PLATYPUS was developed to perform the assignment, provide a measure of reliability of the assignment, and determine the susceptibility tensor anisotropy.

Measurement of dipolar cross-correlation in methylene groups in uniformly 13C-, 15N-labeled proteins.

A CC(CO)NH TOCSY-based 3D pulse scheme is presented for measuring (1)H-(13)C dipole-dipole cross-correlated relaxation at CH(2) positions in uniformly (13)C-, (15)N-labeled proteins. Simulations based on magnetization evolution under relaxation and scalar coupling interactions show that cross-correlation rates between (1)H-(13)C dipoles in CH(2) groups can be simply obtained from the intensities of (13)C triplets. The normalized cross-correlation relaxation rates are related to cross-correlation order parameters for macromolecules undergoing isotropic motion, which reflect the degrees of spatial restriction of CH(2) groups. The study on human intestinal fatty acid binding protein (131 residues) in the presence of oleic acid demonstrates that side chain dynamics at most CH(2) positions can be characterized for proteins less than 15 kDa in size, with the proposed TOCSY-based approach.

Monitoring the early steps of unfolding of dicalcium and mono-Ce3+-substituted forms of P43M calbindin D9k.

Early steps of unfolding of P43M Calbindin D(9k) have been evaluated by NMR spectroscopy on the native dicalcium and on the paramagnetic monocerium-substituted derivative. Although at 2 M GdmHCl the protein core maintains its overall folding and structure, amide (15)N R(2) measurements and cross correlation rates between N-H dipole-dipole relaxation and (15)N CSA relaxation reveal a closer and stronger packing of the hydrophobic interactions in the protein as a response to the presence of denaturing agents in solution. A complete reorientation of the Met43 side chain toward the hydrophobic core is accomplished by the disappearance of the millisecond dynamics observed on the native form of Calbindin D(9k), while cross correlation rates provide evidence that the two-way hydrogen bond between Leu23 and Val61 is broken or substantially weakened. The substitution of the calcium ion in site II with the paramagnetic Ce(3+) ion allowed us to obtain a number of long-range nonconventional constraints, namely, pseudocontact shifts, which were used, together with the NOEs collected on the native state, to monitor subtle structural variations occurring in the non-native state of the protein. Although the average rmsd between the structures of native and non-native states is small (0.48 A), structural rearrangements could be reliably identified. Our results provide unprecedented information about the behavior of Calbindin D(9k) during the early steps of unfolding. Furthermore, they constitute strong evidence of the efficiency of paramagnetism-based constraints in monitoring subtle structural changes that are beyond the sensitivity of an approach based only on NOE.

Lanthanoid endohedral metallofullerenols for MRI contrast agents.

Water-soluble multi-hydroxyl lanthanoid (La, Ce, Gd, Dy, and Er) endohedral metallofullerenes (metallofullerenols, M@C(82)(OH)(n)()) have been synthesized and characterized for the use of magnetic resonance imaging (MRI) contrast agents. The observed longitudinal and transverse relaxivities for water protons, r(1) and r(2), of the metallofullerenols are in the range 0.8-73 and 1.2-80 (sec(-1)mM(-1)), respectively, which are significantly higher than those of the corresponding lanthanoid-DTPA chelate complexes. Among these Gd-metallofullerenols, Gd@C(82)(OH)(n)() has exhibited the highest r(1) and r(2) values in consistent with our previous results. The observed large r(1) of the current metallofullerenols can mainly be ascribed to the dipole-dipole relaxation together with a substantial decrease of the overall molecular rotational motion. The large r(2), except for the Gd-metallofullerenols, have been attributed to the so-called Curie spin relaxation. The MRI phantom studies are also performed and are consistent with these results. The metallofullerenols will be an ideal model for future MRI contrast agents with higher proton relaxivities.

1 H and 205 Tl Spin-Lattice Relaxation at Low Temperatures in Tl 3 H(SO 4 ) 2

It was found from the temperature dependence of spin-lattice relaxation rates of 1 H and 205 Tl nuclei ( 1 H- T 1 m 1 and 205 Tl- T 1 m 1 ) that spin-lattice relaxations of 1 H and 205 Tl result from a magnetic dipole-dipole relaxation. It was also found from the analysis of T 1 's that the energy for the flip-flop motion of protons from O…H--O to O--H…O becomes 2.0 meV at low temperatures. These results suggest that Tl 3 H(SO 4 ) 2 shows the quantum mechanical process such as proton tunneling at low temperatures. The anomalous behavior of 1 H- T 1 m 1 at around 61 K indicates that proton dynamics is changed at around 61 K. Furthermore, it is suggested from the anomalies of 1 H- T 1 m 1 and 205 Tl- T 1 m 1 at around 7 K that Tl 3 H(SO 4 ) 2 shows a phase transition accompanied by the anomaly of the magnetic dipole-dipole fluctuation at around 7 K.

An NMR experiment for the accurate measurement of heteronuclear spin-lock relaxation rates.

Rotating-frame relaxation rates, R(1)(rho), are often measured in NMR studies of protein dynamics. We show here that large systematic errors can be introduced into measured values of heteronuclear R(1)(rho) rates using schemes which are usually employed to suppress cross-correlation between dipole-dipole and CSA relaxation mechanisms. For example, in a scalar-coupled two-spin X-H spin system the use of (1)H WALTZ16 decoupling or (1)H pulses applied at regularly spaced intervals leads to a significant overestimation of heteronuclear R(1)(rho) values. The problem is studied experimentally and theoretically for (15)N-(1)H and (13)C-(1)H spin pairs, and simple schemes are described which eliminate the artifacts. The approaches suggested are essential replacements of existing methodology if accurate dynamics parameters are to be extracted from spin-lock relaxation data sets.

Determination of (1)H homonuclear scalar couplings in unlabeled carbohydrates.

The scarcity of structural information on carbohydrates results from combined difficulties to crystallize and the limitations in NMR analysis. Current methods for determining basic NMR parameters such as (1)H homonuclear scalar couplings are very limited, especially for large molecules such as polysaccharides, oligonucleotides, and the carbohydrate part of glycoproteins. In this paper, a NMR experiment for the determination of endocyclic (1)H homonuclear scalar couplings ((3)J(HH)) in unlabeled carbohydrates is presented. In addition to scalar couplings, cross-correlated dipole-dipole relaxation rates were measured for large polysaccharides. The measurement of all endocyclic homonuclear coupling constants within monosaccharide units is presented for lactose, a model disaccharide, and for a natural-abundance 2 MDa bacterial polysaccharide excreted by Streptococcus thermophilus Sfi39.

Characterization of the [Ag(dmb)(2)(+)](n) oligomers (dmb = 1,8-diisocyano-p-menthane) in solution.

The crystallographically characterized polymers [[Ag(dmb)(2)]Y](n) (Y = BF(4)(-), NO(3)(-), ClO(4)(-)) extensively dissociate in solution, contrarily to the Cu analogue, and common molecular weight determination techniques such as light scattering, osmometric, and intrinsic viscosity measurements fail to provide data allowing full characterization. Using pulsed NMR experiments, notably (13)C NMR T(1) (spin lattice relaxation time) and NOE (nuclear Overhauser enhancement) measurements on various ionic [[Ag(dmb)(2)]Y](n) materials (Y = BF(4)(-), NO(3)(-), ClO(4)(-)) and their related mononuclear [Ag (CN-t-Bu)(4)]Y salts in acetonitrile-d(3) (as comparative standards), the dipole-dipole spin lattice relaxation times (T(1)(DD)) of a selected quaternary (13)C probe are measured. These data allow us to extract the correlation times (tau(c)), which in turn permit us to estimate the volume of the tumbling species in solution. The comparison of the data between the [Ag(dmb)(2)(+)](n) and Ag(CN-t-Bu)(4)(+) species indicates the oligomeric nature of the former species, where the average number of Ag(dmb)(2)(+) approximately 8 (M(n) approximately 4000-5000).

A 15N NMR mobility study on the dicalcium P43M calbindin D9k and its mono-La3+-substituted form.

Calbindin D(9k) is a dicalcium binding protein consisting of two helix-loop-helix EF-hand motifs joined together by a flexible linker region where one metal ion can bind to each of the two loops. A proline residue at position 43 in the linker region displays cis-trans isomerism in the wild-type (WT) protein. Such isomerism appeared to be removed by substituting the proline with a glycine or a methionine in the P43G or P43M mutant. We have extended the available mobility studies on the P43M mutant through amide (15)N R(1), R(2), and R(1)(rho)() measurements. This has revealed unexpected conformational equilibria on the millisecond time scale involving residues 38, 42-44, and 46 in the linker region and residues 18 and 19 in calcium binding site I with similar energy barriers. These data are discussed in comparison with those available for the WT, as well as the apo-, mono-, and disubstituted P43G mutant. Quantification of water-amide proton exchange rates using saturation transfer and qualitative application of (15)N-(CLEANEX-PM)-FHSQC shows the values are in agreement with high mobility for the above-mentioned residues. Cross correlation between N-H dipole-dipole relaxation and (15)N CSA relaxation indicates that some of these mobility differences may extend to the sub-nanosecond time scale. Similar data were also obtained for the derivative where the calcium ion in the C-terminal loop was replaced with lanthanum. The results presented here show that, contrary to expectations, there are significant differences in dynamics between the dicalcium state of P43G and P43M and that these differences are not confined to the flexible linker region containing the point mutation. They also demonstrate that substitution of a lanthanide ion for calcium, which is a common procedure, does not significantly alter the mobility of the native protein.

Nuclear Overhauser Enhancement Spectroscopy Cross-Relaxation Rates and Ethanol Distribution across Membranes

Measurement of nuclear Overhauser enhancement spectroscopy cross-relaxation rates between ethanol and palmitoyloleoylphosphatidylcholine bilayers was combined with atomic-level molecular dynamics simulations. The molecular dynamics trajectories yielded autocorrelation functions of proton dipole-dipole interactions, and, consequently, relaxation times and cross-relaxation rates. These analyses allow the measured cross-relaxation rates to be interpreted in terms of relative interaction strengths with the various segments of the lipid molecule. We determined that cross-relaxation between ethanol and specific lipid resonances is primarily determined by the sites of interaction with some modulation due to lipid disorder and to local differences in intramolecular lipid dynamics. The rates scale linearly with the lifetime of temporary ethanol-lipid associations. Ethanol interacts with palmitoyloleoylphosphatidylcholine bilayers primarily via hydrophilic interactions, in particular the formation of hydrogen bonds to the lipid phosphate group. There is a weak contribution to binding from hydrophobic interaction with lipid chain segments near the glycerol. However, the strength of hydrophobic interactions is insufficient to compensate for the energetic loss of locating ethanol in an exclusively hydrophobic environment, resulting in a probability of locating ethanol in the bilayer center that is three orders of magnitude lower than locating ethanol at the lipid/water interface. The low cross-relaxation rates between terminal methyl protons of hydrocarbon chains and ethanol are as much the result of infrequent chain upturns as of brief excursions of ethanol into the region of lipid hydrocarbon chains near the glycerol. The combination of nuclear magnetic resonance measurements and molecular dynamics simulations offers a general pathway to study the interaction of small molecules with the lipid matrix at atomic resolution.

Cross correlation rates between Curie spin and dipole-dipole relaxation in paramagnetic proteins: the case of cerium substituted calbindin D9k.

Cross correlation rates between Curie spin relaxation and H-N dipole-dipole coupling (gamma(HM,HN)CS,DD) have been determined for a calcium binding protein, Calbindin D9k, in which one of the two calcium ions is substituted with cerium(III). Gamma(HM,HN)CS,DD values depend on both the metal-to-proton distances and the M-H-N angles and can be used as an additional constraint in order to refine the solution structure of paramagnetic metalloproteins. For this purpose, we have implemented a new module (CCR-DYANA) in a version of the program DYANA (PARAMAGNETIC-DYANA), which can be used together with other paramagnetism-based constraints such as pseudocontact shifts, residual dipolar couplings and hyperfine based Karplus relationships. This integrated structure calculation protocol has the advantage that different paramagnetic-based constraints are treated by the same algorithm in a way that the efficiency of each class of constraints can be analyzed and compared.

Determination of the glycosidic torsion angles in uniformly 13C-labeled nucleic acids from vicinal coupling constants 3J(C2)/4-H1' and 3J(C6)/8-H1'.

A two-dimensional, quantitative J-correlation NMR experiment for precise measurements of the proton-carbon vicinal coupling constants 3J(C2)/4-H1' and 3J(C6)/8-H1' in uniformly 13C-labeled nucleic acids is presented. To reduce loss of signal due to 1H-13C dipole-dipole relaxation, a multiple-quantum constant time experiment with appropriately incorporated band selective 1H and 13C pulses was applied. The experiment is used to obtain the 3J(C2)/4-H1' and 3J(C6)/8-H1' coupling constants in a uniformly 13C, 15N-labeled [d(G4T4G4)]2 quadruplex. The measured values and glycosidic torsion angles in the G-quadruplex, obtained by restrained molecular dynamics with explicit solvent using the previously published restraints, along with selected data from the literature are used to check and modify existing parameters of the Karplus equations. The parameterizations obtained using glycosidic torsion angles derived from the original solution and recently determined X-ray structures are also compared.

NMR EVIDENCE OF HYDROGEN BOND IN 1-ETHYL-3-METHYLIMIDAZOLIUM-TETRAFLUOROBORATE ROOM TEMPERATURE IONIC LIQUID

The 1-ethyl-3-methylimidazolium-tetrafluoroborate (EMI-BF4) room-temperature molten salt was investigated with NMR techniques. Diffusion coefficients measured at temperatures ranging from 300 to 360 K indicate phase-change occurred in the vicinity of 333 K, which is supported by 11B quadrupolar relaxation rates. Combined with the sizes of diffusion particles obtained form the Stokes-Einstein equation, this phase change is ascribed to that the diffusion particle is transformed from “discrete ion-pair” to “individual ion” at temperatures above 335 K due to decomposition of the EMI-BF4 ion pair. Analysis of the 13C dipole-dipole relaxation rates identifies the formation of hydrogen bond(C2H…F) between the counter ions, EMI+ and BF4 −. That the values of viscosity in EMI-BF4 are higher than those in EMI-AlCl4 at corresponding temperatures is therefore understood according to the viscosity approach employed to investigate the H-bonding in molten salts

Transient13C–1H Nuclear Overhauser Effect in Liquid Crystal

Significant transient 13 C– 1 H nuclear Overhauser effect (NOE) is observed in liquid crystal, and is explained by a local 13 C– 1 H dipole-dipole interaction and relaxation. Solomon's classical two-pulse experiment gives a significant maximum in signal intensity of 2.5 times the equilibrium magnetization, and also gives a way of estimating zero-quantum transition probability for a dipole-coupled 13 C– 1 H pair. The conventional inversion recovery sequence is recommended for the experimental determination of 13 C spin-lattice relaxation rates. Magnetization recovery after the Hartmann-Hahn signal enhancement deviates strongly from exponential behavior. Experimental data are shown for an antiferroelectric chiral smectic liquid crystal MHPOBC.

The secondary structure of a membrane-modifying peptide in a supramolecular assembly studied by PELDOR and CW-ESR spectroscopies.

The new technique of pulsed electron-electron double resonance in electron spin-echo (PELDOR) in combination with the CW-ESR method has been used to investigate the secondary structure of a double spin-labeled peptide (the [TOAC-1,8]-analogue of the peptaibol antibiotic trichogin GA IV) that is hidden into a tetrameric supramolecular assembly of unlabeled peptide molecules. The magnetic dipole-dipole relaxation of spin labels has been experimentally studied in glassy solutions of the double-labeled peptide frozen to 77 K in a mixture of chloroform-toluene with an excess of unlabeled peptide. The PELDOR signal oscillations have been observed at high degrees of dilution with unlabeled peptide. The intramolecular distance between the spin labels of the peptide molecule in the aggregate has been determined from the oscillation frequency to be 15.7 A which is close to the value of (approximately equal to) 14 A calculated for a 3(10)-helical structure. Estimation of the fraction of this ordered secondary structure shows that about 19% of the peptide molecules in aggregates are folded in the 3(10)-helical conformation. The present experimental results are consistent with our molecular model presented in J. Am. Chem. Soc. 2000, 122, 3843-3848, wherein four amphiphilic 3(10)-helical peptide molecules form a vesicular system with the polar amino acid side chains pointing to the interior, and the apolar side chains, to the exterior of the cluster. The experimental data were compared with the results obtained with other techniques.

THE NMR CONFORMATION STUDY OF THE COMPLEXES OF DEOXYCYTIDINE KINASE (dCK) AND 2′-DEOXYCYTIDINE/2′-DEOXYADENOSINE

The structures of the bound 13C/2H double-labelled 2′(R/S), 5′(R/S)-2H2-1′,2′,3′,4′,5′-13C5-2′-deoxyadenosine and the corresponding 2′-deoxycytidine moieties in the complexes with human deoxycytidine kinase (dCK) have been characterized for the first time by the solution NMR spectroscopy, using Transferred Dipole-Dipole Cross-correlated Relaxation and Transferred nOe experiments. It has been shown that the ligand adopts a South-type sugar conformation when bound to dCK.

Separation of anisotropy and exchange broadening using (15)N CSA-(15)N-(1)H dipole-dipole relaxation cross-correlation experiments.

Based on the measurement of cross-correlation rates between (15)N CSA and (15)N-(1)H dipole-dipole relaxation we propose a procedure for separating exchange contributions to transverse relaxation rates (R(2) = 1/T(2)) from effects caused by anisotropic rotational diffusion of the protein molecule. This approach determines the influence of anisotropy and chemical exchange processes independently and therefore circumvents difficulties associated with the currently standard use of T(1)/T(2) ratios to determine the rotational diffusion tensor. We find from computer simulations that, in the presence of even small amounts of internal flexibility, fitting T(1)/T(2) ratios tends to underestimate the anisotropy of overall tumbling. An additional problem exists when the N-H bond vector directions are not distributed homogeneously over the surface of a unit sphere, such as in helix bundles or beta-sheets. Such a case was found in segment 4 of the gelation factor (ABP 120), an F-actin cross-linking protein, in which the diffusion tensor cannot be calculated from T(1)/T(2) ratios. The (15)N CSA tensor of the residues for this beta-sheet protein was found to vary even within secondary structure elements. The use of a common value for the whole protein molecule therefore might be an oversimplification. Using our approach it is immediately apparent that no exchange broadening exists for segment 4 although strongly reduced T(2) relaxation times for several residues could be mistaken as indications for exchange processes.