Investigations of polymer dynamics in nanoporous media by field cycling NMR relaxometry and the dipolar correlation effect

The chain dynamics of short-chain perfluoropolyether melts confined in Vycor nanoporous media has been characterized by field cycling nuclear magnetic resonance relaxometry and the dipolar correlation effect. The slowdown of motions under confinement, leading to larger residual dipolar couplings, has been probed by looking at the quotient of stimulated and primary echoes. Using field cycling relaxometry, it has been shown that there is strong evidence of reptation-like motion, even for such short-chain polymers as shown by the frequency and molecular weight dependences of the spin-lattice relaxation time.     

A simulation algorithm based on Bloch equations and product operator matrix: application to dipolar and scalar couplings

A product operator matrix is proposed to describe scalar couplings in liquid NMR. Combination of the product operator matrix and non-linear Bloch equations is employed to describe effects of chemical shift, translational diffusion, dipolar field, radiation damping, and relaxation in multiple spin systems with both scalar and dipolar couplings. A new simulation algorithm based on this approach is used to simulate NMR signals from dipolar field effects in the presence of scalar couplings. Several typical coupled spin systems with both intra-molecular scalar couplings and inter-molecular dipolar couplings are simulated. Monte Carlo methods are incorporated into simulations as well to analyze diffusion process in these complicated spin systems. The simulated results of diffusion and relaxation parameters and 2D NMR spectra are coincident with the experimental measurements, and agree with theoretical predictions as well. The simulation algorithm presented herein therefore provides a convenient means for designing pulse sequences and quantifying experimental results in complex coupled spin systems.     

Magnetization Transfer via Residual Dipolar Couplings: Application to Proton–Proton Correlations in Partially Aligned Proteins

A novel three-dimensional NMR experiment is reported that allows the observation of correlations between amide and other protons via residual dipolar couplings in partially oriented proteins. The experiment is designed to permit quantitative measurement of the magnitude of proton–proton residual dipolar couplings in larger molecules and at higher degree of alignments. The observed couplings contain data valuable for protein resonance assignment, local protein structure refinement, and determination of low-resolution protein folds.     

Two Simple NMR Experiments for Measuring Dipolar Couplings in Asparagine and Glutamine Side Chains

Residual dipolar couplings are now widely used for structure determination of biological macromolecules. Until recently, the main focus has been on measurement of dipolar couplings in the protein main chain. However, with the aim of more complete protein structure, it is also essential to have information on the orientation of protein side chains. In addition, residual dipolar couplings can potentially be employed to study molecular dynamics. In this Communication, two simple NH(2) and spin-state edited experiments are presented for rapid and convenient determination of five residual dipolar couplings from (15)N, (1)H correlation spectrum in asparagine and glutamine side chains. The pulse sequences are demonstrated on two proteins, 30.4-kDa Cel6A in diluted liquid crystal phase and 18-kDa human cardiac troponin C in water.     

Magnetization transfer via residual dipolar couplings: application to proton-proton correlations in partially aligned proteins

A novel three-dimensional NMR experiment is reported that allows the observation of correlations between amide and other protons via residual dipolar couplings in partially oriented proteins. The experiment is designed to permit quantitative measurement of the magnitude of proton-proton residual dipolar couplings in larger molecules and at higher degree of alignments. The observed couplings contain data valuable for protein resonance assignment, local protein structure refinement, and determination of low-resolution protein folds.     

Proton residual dipolar couplings by NMR magnetization exchange in cross-linked elastomers: determination and imaging.

Proton nuclear magnetic resonance (NMR) magnetization exchange is used to investigate residual dipolar couplings in a series of cross-linked poly(styrene-cobutadiene) elastomers. A new model for the dipolar unit is used for the evaluation of the signal decay in magnetization exchange experiments. It takes into account an extended residual dipolar coupling network along the polymer chain. It is shown that in the regime of short mixing times, information about the residual dipolar coupling between methine and methylene protons can be obtained which is not affected by other inter- and intramolecular dipolar couplings. The dynamic order parameter of methine-methylene protons is measured and correlated with cross-link density. This study certifies the quality of a filter for magnetization from residual dipolar couplings which exploit magnetization exchange. The filter can be used to generate contrast in NMR images of heterogeneous elastomers. The first proton NMR parameter image of a dynamic order parameter is presented for a phantom made from poly(styrene-cobutadiene) samples with different cross-link densities.     

Measurement of one and two bond N–C couplings in large proteins by TROSY-based J-modulation experiments

Residual dipolar couplings (RDCs) between NC′ and NC^α atoms in polypeptide backbones of proteins contain information on the orientation of bond vectors that is complementary to that contained in NH RDCs. The ¹J_NC^α and ²J_NC^α scalar couplings between these atoms also display a Karplus relation with the backbone torsion angles and report on secondary structure. However, these N–C couplings tend to be small and they are frequently unresolvable in frequency domain spectra having the broad lines characteristic of large proteins. Here a TROSY-based J-modulated approach for the measurement of small ¹⁵N–¹³C couplings in large proteins is described. The cross-correlation interference effects inherent in TROSY methods improve resolution and signal to noise ratios for large proteins, and the use of J-modulation to encode couplings eliminates the need to remove frequency distortions from overlapping peaks during data analysis. The utility of the method is demonstrated by measurement of ¹J_NC′, ¹J_NC^α, and ²J_NC^α scalar couplings and ¹D_NC′ and ¹D_NC^α residual dipolar couplings for the myristoylated yeast ARF1·GTPγs protein bound to small lipid bicelles, a system with an effective molecule weight of 70 kDa.     

Field-dependent nuclear relaxation of spins 1/2 induced by dipole-dipole couplings to quadrupole spins: LaF3 crystals as an example

A general theory of spin-lattice nuclear relaxation of spins I=1/2 caused by dipole-dipole couplings to quadrupole spins S1, characterized by a non-zero averaged (static) quadrupole coupling, is presented. In multispin systems containing quadrupolar and dipolar nuclei, transitions of spins 1/2 leading to their relaxation are associated through dipole-dipole couplings with certain transitions of quadrupole spins. The averaged quadrupole coupling attributes to the energy level structure of the quadrupole spin and influences in this manner relaxation processes of the spin 1/2. Typically, quadrupole spins exhibit also a complex multiexponential relaxation sensed by the dipolar spin as an additional modulation of the mutual dipole-dipole coupling. The proposed model includes both effects and is valid for an arbitrary magnetic field and an arbitrary quadrupole spin quantum number. The theory is applied to interpret fluorine relaxation profiles in LaF3 ionic crystals. The obtained results are compared with predictions of the 'classical' Solomon relaxation theory.     

残留偶极耦合及其在蛋白质结构研究中的应用

近年来溶液中残留偶极耦合常数被用来获取生物大分子化学键之间相对取向等长程构象约束条件,用于计算或优化蛋白质及其复合物的三维空间结构. 介绍了用异核多维NMR技术测量残留偶极耦合常数的方法,及其在蛋白质结构计算中的一些应用：优化蛋白质溶液结构,评价蛋白质结构质量,确定蛋白质结构域取向,获取有关配体的构象和取向的信息,在缺乏NOE数据时构建蛋白质结构等.      

Protein Backbone N Relaxation Rates as a Tool for the Diagnosis of Structure Quality

In the work reported herein we define a structure validation factor that depends on protein backbone ¹⁵N relaxation rates. This is an alternative method to the previously defined quality factors derived from anisotropic chemical shifts or residual dipolar couplings. We have used the structure dependence of ¹⁵N relaxation rates of anisotropically tumbling proteins to calculate this structure diagnosis factor and have used it to demonstrate the improvement of protein structures refined with residual dipolar couplings.     

Spatial structure of triglycine determined by the residual dipolar coupling analysis

The possibility to determine the relatively small organic compound conformations by the approach on the basis of the analysis of the residual dipolar couplings¹H−¹³C in the molecules partially aligned in lyotropic liquid crystalline media has been considered. This approach has been used in the nuclear magnetic resonance investigation of the triglycine structure in lyotropic medium (cetylpyridinium bromide/n-hexanol). The conformation of triglycine in solution has been established astrans-trans on the basis of the experimental data of observed couplings.     

Measurement of Dipolar Cross Correlation in Scalar-Coupled Systems

In systems with dipolar relaxation in isotropic phase, it is possible to measure the extent of cross correlation of the fluctuations of two selected dipole–dipole interactions A–M and A–X by selectively exciting and spin-locking the transverse magnetization of spin A. If the system comprises only three spins A, M, and X, the conversion of in-phase magnetization[formula]into doubly antiphase magnetization[formula]during the spin-locking period occurs spontaneously through relaxation. The rate of this conversion is proportional to the spectral density of the cross correlation of the random fluctuations of the dipolar A–M and A–X interactions. In this paper, larger systems, comprising at least a fourth spin K, are investigated. The complexity of the situation is increased, since other forms of three-spin order such as[formula]or[formula]become accessible. Furthermore, this paper addresses the role of scalar couplings, which are a prerequisite for making three-spin order observable, but which are also a source of perturbations, since scalar couplings can contribute significantly to the creation of various three-spin-order terms. If the spin-locking field is too weak compared to the width of the multiplet under investigation, residual scalar interactions lead to the generation of three-spin order. If the spin-locking field is too strong compared to the relative offsets of other “passive” spins, further complications occur. These can be avoided most effectively by using very high static magnetic fields. If coherent contributions to three-spin order can be suppressed or accounted for through simulations, the remaining buildup of three-spin-order terms arising from dipolar cross-correlation effects can be interpreted in terms of structural and motional parameters.     

Quantification of homonuclear dipolar coupling networks from magic-angle spinning 1H NMR

Numerical simulations of magic-angle spinning (MAS) spectra of dipolar-coupled nuclear spins have been used to assess different approaches to the quantification of dipolar couplings from ¹H solid-state NMR. Exploiting the translational symmetry of periodic spin systems allows extended networks with ‘realistic’ numbers of spins to be considered. The experimentally accessible parameter is shown to be the root-sum-square of the dipolar couplings to a given spin. The effectiveness of either fitting the resulting spinning sideband spectra to small spin system models, or using analyses based on moment expansions, has been examined. Fitting of the spinning sideband pattern is found to be considerably more robust with respect to experimental noise than frequency domain moment analysis. The influence of the MAS rate and system geometry on robustness of the quantification is analysed and discussed.     

Variation of molecular alignment as a means of resolving orientational ambiguities in protein structures from dipolar couplings

Residual dipolar couplings for pairs of proximate magnetic nuclei in macromolecules can easily be measured using high-resolution NMR methods when the molecules are dissolved in dilute liquid crystalline media. The resulting couplings can in principle be used to constrain the relative orientation of molecular fragments in macromolecular systems to build a complete structure. However, determination of relative fragment orientations based on a single set of residual dipolar couplings is inherently hindered by the multi-valued nature of the angular dependence of the dipolar interaction. Even with unlimited dipolar data, this gives rise to a fourfold degeneracy in fragment orientations. In this Communication, we demonstrate a procedure based on an order tensor analysis that completely removes this degeneracy by combining residual dipolar coupling measurements from two alignment media. Application is demonstrated on (15)N-(1)H residual dipolar coupling data acquired on the protein zinc rubredoxin from Clostridium pasteurianum dissolved in two different bicelle media.     

Sensitivity-enhanced 2D IPAP,TROSY-anti-TROSY,and E.COSY experiments: alternatives for measuring dipolar 15N-1HN couplings

Sensitivity-enhanced versions of the IPAP, TROSY-anti-TROSY, and E.COSY experiments for measuring one-bond 15N-1HN couplings are presented. Together with the previously developed sensitivity-enhanced E.COSY-type HSQC experiment they comprise a suite of sensitivity-enhanced experiments that allows one to chose the optimal spectrum for accurate measurement of one-bond 15N-1HN residual dipolar couplings in proteins. Since one-bond 15N-1HN residual dipolar couplings play uniquely important roles in structural NMR, these additional methods provide further tools for improving structure determination of proteins and other biological macromolecules.     

Relaxation times and line widths of isotopically-substituted nitroxides in aqueous solution at X-band

Optimization of nitroxides as probes for EPR imaging requires detailed understanding of spectral properties. Spin lattice relaxation times, spin packet line widths, nuclear hyperfine splitting, and overall lineshapes were characterized for six low molecular weight nitroxides in dilute deoxygenated aqueous solution at X-band. The nitroxides included 6-member, unsaturated 5-member, or saturated 5-member rings, most of which were isotopically labeled. The spectra are near the fast tumbling limit with T₁∼T₂ in the range of 0.50–1.1 μs at ambient temperature. Both spin–lattice relaxation T₁ and spin–spin relaxation T₂ are longer for ¹⁵N- than for ¹⁴N-nitroxides. The dominant contributions to T₁ are modulation of nitrogen hyperfine anisotropy and spin rotation. Dependence of T₁ on nitrogen nuclear spin state m_I was observed for both ¹⁴N and ¹⁵N. Unresolved hydrogen/deuterium hyperfine couplings dominate overall line widths. Lineshapes were simulated by including all nuclear hyperfine couplings and spin packet line widths that agreed with values obtained by electron spin echo. Line widths and relaxation times are predicted to be about the same at 250 MHz as at X-band.     

Variable angle NMR spectroscopy and its application to the measurement of residual chemical shift anisotropy

The successful measurement of anisotropic NMR parameters like residual dipolar couplings (RDCs), residual quadrupolar couplings (RQCs), or residual chemical shift anisotropy (RCSA) involves the partial alignment of solute molecules in an alignment medium. To avoid any influence of the change of environment from the isotropic to the anisotropic sample, the measurement of both datasets with a single sample is highly desirable. Here, we introduce the scaling of alignment for mechanically stretched polymer gels by varying the angle of the director of alignment relative to the static magnetic field, which we call variable angle NMR spectroscopy (VA-NMR). The technique is closely related to variable angle sample spinning NMR spectroscopy (VASS-NMR) of liquid crystalline samples, but due to the mechanical fixation of the director of alignment no sample spinning is necessary. Also, in contrast to VASS-NMR, VA-NMR works for the full range of sample inclinations between 0° and 90°. Isotropic spectra are obtained at the magic angle. As a demonstration of the approach we measure 13C-RCSA values for strychnine in a stretched PDMS/CDCl? gel and show their usefulness for assignment purposes. In this context special care has been taken with respect to the exact calibration of chemical shift data, for which three approaches have been derived and tested.     

蛋白质顺磁标记技术与生物核磁共振中的赝接触位移

未配对电子与蛋白质分子自旋核的作用能提供丰富的长程结构信息,这些顺磁信息通常可用顺磁弛豫增强、赝接触位移和残余偶极耦合描述,其中赝接触位移包含生物大分子内重要的距离和角度信息.稀土离子具有相似的配位化学性质和不同的顺磁物理特性,而大多稀土离子具有磁各向异性,在与大分子作用过程中会产生赝接触位移.由于大多数蛋白质没有顺磁中心,获得这些顺磁信息需要通过定点选择标记蛋白质来实现.该文旨在对近年来蛋白质顺磁标记的方法和进展进行介绍,在顺磁标记基础上阐述赝接触位移在结构生物学中的应用.     

P.E.HSQC: a simple experiment for simultaneous and sign-sensitive measurement of (1JCH+DCH) and (2JHH+DHH) couplings

The angular information content of residual dipolar couplings between nuclei of fixed distance makes the accurate and sign-sensitive measurement of (¹J_CH + D_CH) and (²J_HH + D_HH) couplings highly desirable. Experiments published so far are typically highly specialized for the effective measurement of a subset of couplings. The P.E.HSQC presented here, is an E.COSY based experiment which allows the simultaneous measurement of all heteronuclear and homonuclear couplings within CH, CH₂, and CH₃ groups in a single spectrum with the necessary precision and sign information. The simplicity of the approach and the absence of artefacts like phase distortions due to antiphase evolution make it ideally suited for coupling determination of organic molecules at natural abundance.     

Quantum Treatment of Intermolecular Multiple-Quantum Coherences with IntramolecularJCoupling in Solution NMR

A recently introduced density matrix picture for dipolar effects in solution NMR (1996,J. Chem. Phys.105,874) gave complete solutions for intermolecular multiple-quantum coherences for single-component samples without scalar couplings. This paper, for the first time, shows that this quantum picture can lead to explicit signal expressions for multicomponent samples of molecules with internal scalar couplings (here assumed to generate a first-order spectrum) and long-range dipolar couplings. Experimental observation of a triplet in the indirectly detected dimension for a heteronuclear CRAZED sequence (¹³CHCl₃sample, ZQ or 2Q coherences) gives clear evidence that the coupling is due to the intermolecular dipolar coupling. We also make comparisons with classical pictures which introduce the dipolar demagnetization field in multicomponent spin systems.