Strategies for measurements of pseudocontact shifts in protein NMR spectroscopy.

Paramagnetic metal ions bound to proteins generate a dipolar field that can be accurately probed by pseudocontact shifts (PCS) displayed by the protein's nuclear spins. PCS are highly useful for determining the coordinates of individual spins in the molecule and for rapid structure determinations of entire protein-protein and protein-ligand complexes. However, PCS measurements require reliable resonance assignments for the molecule in its paramagnetic state and in a diamagnetic reference state. This article discusses different approaches for pairwise resonance assignments, with emphasis on a strategy which exploits chemical exchange between the two states.     

Weak alignment of biomacromolecules in collagen gels: an alternative way to yield residual dipolar couplings for NMR measurements

Collagen, consisting of glycine, proline, and hydroxyproline, is a fibrous protein that can form a rope-like left-hand triple helix structure. It is demonstrated here that the collagen gels prepared from polymerization in the magnetic field can provide weak alignment for protein. The alignment order induced by collagen gels is quite small when compared to other alignment media, but the magnitude of the dipolar couplings can be easily scaled up by increasing the initial concentration of collagen. The collagen gels showed good pH and detergent tolerance. These advantages of collagen gels make it a promising candidate for the alignment of large biomolecules or membrane protein-detergent complexes in the magnetic field.     

Lanthanide-binding peptides for NMR measurements of residual dipolar couplings and paramagnetic effects from multiple angles

Lanthanide-binding peptide tags (LBTs) containing a single cysteine residue can be attached to proteins via a disulfide bond, presenting a flexible means of tagging proteins site-specifically with a lanthanide ion. Here we show that cysteine residues placed in different positions of the LBT can be used to expose the protein to different orientations of the magnetic susceptibility anisotropy (delta chi) tensor and to generate different molecular alignments in a magnetic field. Delta chi tensors determined by nuclear magnetic resonance (NMR) spectroscopy for LBT complexes with Yb3+, Tm3+, and Er3+ suggest a rational way of producing alignment tensors with different orientations. In addition, knowledge of the delta chi tensor of LBT allows modeling of the protein-LBT structures. Despite evidence for residual mobility of the LBTs with respect to the protein, the pseudocontact shifts and residual dipolar couplings displayed by proteins disulfide-bonded to LBTs are greater than those achievable with most other lanthanide binding tags.     

On the use of pseudocontact shifts in the structure determination of metalloproteins

The utility of pseudocontact shifts in the structure refinement of metalloproteins has been evaluated using a native, paramagnetic Cu(2+) metalloprotein, plastocyanin from Anabaena variabilis (A.v.), as a model protein. First, the possibility of detecting signals of nuclei spatially close to the paramagnetic metal ion is investigated using the WEFT pulse sequence in combination with the conventional TOCSY and (1)H-(15)N HSQC sequences. Second, the importance of the electrical charge of the metal ion for the determination of correct pseudocontact shifts from the obtained chemical shifts is evaluated. Thus, using both the Cu(+) plastocyanin and Cd(2+)-substituted plastocyanin as the diamagnetic references, it is found that the Cd(2+)-substituted protein with the same electrical charge of the metal ion as the paramagnetic Cu(2+) plastocyanin provides the most appropriate diamagnetic reference signals. Third, it is found that reliable pseudocontact shifts cannot be obtained from the chemical shifts of the (15)N nuclei in plastocyanin, most likely because these shifts are highly dependent on even minor differences in the structure of the paramagnetic and diamagnetic proteins. Finally, the quality of the obtained (1)H pseudocontact shifts, as well as the possibility of improving the accuracy of the obtained structure, is demonstrated by incorporating the shifts as restraints in a refinement of the solution structure of A.v. plastocyanin. It is found that incorporation of the pseudocontact shifts enhances the precision of the structure in regions with only few NOE restraints and improves the accuracy of the overall structure.     

Rapid measurement of pseudocontact shifts in metalloproteins by proton-detected solid-state NMR spectroscopy

Pseudocontact shifts (PCSs) arise in paramagnetic systems in which the susceptibility tensor is anisotropic. PCSs depend upon the distance from the paramagnetic center and the position relative to the susceptibility tensor, and they can be used as structural restraints in protein structure determination. We show that the use of (1)H-detected solid-state correlations provides facile and rapid detection and assignment of site-specific PCSs, including resolved (1)H PCSs, in a large metalloprotein, Co(2+)-substituted superoxide dismutase (Co(2+)-SOD). With only 3 mg of sample and a small set of experiments, several hundred PCSs were measured and assigned, and these PCSs were subsequently used in combination with (1)H-(1)H distance and dihedral angle restraints to determine the protein backbone geometry with a precision paralleling those of state-of-the-art liquid-state determinations of diamagnetic proteins, including a well-defined active site.     

镧系离子与磺基水杨酸配合物接触位移和假接触位移的分离

本工作测定了顺磁性镧系离子与磺基水杨酸的配合物上每个^1H的NMR诱导位移, 并利用公式ΔMHL^P/=A+D/·A2^0G进行了线性处理, 根据直线的斜率、截距和D、的理论值对诱导位移实行了接触位移和假接触位移的分离。     

Weak calcium-mediated interactions between Lewis X-related trisaccharides studied by NMR measurements of residual dipolar couplings

The Lewis X (LeX) determinant, a trisaccharide with the carbohydrate sequence Galbeta(1-->4)[Fucalpha(1-->3)]GlcNAcbeta, is believed to be responsible for Ca2+-mediated cell-cell interactions. In partly oriented phases composed of mixtures of penta(ethyleneglycol)monododecyl ether HO(CH2CH2O)5C12H25 and n-hexanol in the presence of Ca2+ ions, the variation of the residual dipolar couplings 1DCH of various CiHi vectors in LeX as a function of the concentration of the trisaccharide demonstrates the existence of very weak LeX-Ca2+-LeX complexes in solution. Synthetic 3-, 4-, and 6-deoxy-LeX variants were also shown to form complexes in the presence of calcium ions, despite the replacement of one of their hydroxyl groups by hydrogen atoms. This is the first direct observation in solution of a calcium-mediated interaction between LeX molecules.     

Theory of NMR shifts and endor spectroscopy in paramagnetic complexes

A theory of NMR and ENDOR spectroscopy in paramagnetic complexes is presented. The relevant matrix elements of both the contact and pseudo-contact interaction within the eigenstates of the magnetic electrons are treated exactly. It is shown, that the interactions depend upon the azimuthal angle ? of the resonant nucleus, even for high point symmetries of the paramagnetic center. In the case of NMR shift measurements, where the spin-lattice relaxation time is short, the interaction of the nuclear spin with the electron's spin and orbital motion can be described by a temperature-dependent induced magnetic field which is not parallel to the applied field. The results differ even in lowest order of the multipole expansion from previous theories. The pseudo-contact shift for rare-earth complexes is correlated to susceptibility in a modified form. In iron-series complexes, such a correlation is generally not possible. Here, the familiar point-dipole approximation used in earlier theories is valid only, if the orbital angular momentum is quenched or if the resonant nucleus lies on the p-fold rotation axis and p ≥ 2.     

A method for correction of matrix effects in activation analysis based on characteristic X-ray measurements

The empirical coefficient method represents the absorption and enhancement effect of each element on each other by parameters independent of mass concentrations. This method is used together with the internal standard method for the determination of cadmium, bromine and selenium by 14 MeV neutron activation followed by X-ray spectrometry. The results of analysis show a relative error not more than ±5%.     

Evidence of 29Si NMR paramagnetic shifts in rare-earth zeolite LSX

Paramagnetic shifts have been observed for the first time in rare-earth zeolites; the 29Si MAS NMR spectra of rare-earth ion-exchanged low silica X show a large range of isotropic chemical shifts that can be attributed to Fermi contact interactions with the lanthanide electronic moments.     

Electron paramagnetic resonance measurements and transient effects of the phosphorescent state of 1, 3-diazaazulene

The zero-field splittings and g-tensors of the phosphorescent state of 1,3-diazaazulene imbedded in the naphthalene crystal have been measured at 77 K. The transient effect of the photoexcitation to the metastable triplet state is observed and the dynamics of the photomagnetization is deduced from the study.     

Cooperative hydrogen bonding effects are key determinants of backbone amide proton chemical shifts in proteins

A computational methodology for backbone amide proton chemical shift (delta(H)) predictions based on ab initio quantum mechanical treatment of part of the protein is presented. The method is used to predict and interpret 13 delta(H) values in protein G and ubiquitin. The predicted amide-amide delta(H) values are within 0.6 ppm of experiment, with a root-mean-square deviation (RMSD) of 0.3 ppm. We show that while the hydrogen bond geometry is the most important delta(H)-determinant, longer-range cooperative effects of extended hydrogen networks make significant contributions to delta(H). We present a simple model that accurately relates the protein structure to delta(H).     

Optimisation of alignment materials for minimising residual retardation in in-plane switching liquid crystal display

The light leakage in a black state of in-plane switching (IPS) liquid crystal display (LCD) associated with rubbing process has been investigated. The mechanical rubbing process with a cloth caused orientation disorders in the liquid crystal directors and these partial orientation disorders result in residual retardations of the IPS LCD, causing the light leakages at the black state. In this study, we theoretically estimated how the light leakage is associated with the rubbing uniformity using 2 × 2 Jones matrix equation and also experimentally confirmed how it is associated with structural properties of the alignment layer. The light leakage was clearly reduced in the alignment layer with reduced crystallinity and flexibility.     

Quantum chemical prediction of the 13C NMR shifts in alkyl and chlorocorannulenes: correction of chlorine effects

Prediction of the ¹³C NMR shifts of sym-pentachlorocorannulene and decachlorocorannulene provided impetus for the development of a correction scheme based on a regression of experimental and quantum chemical data. A training set of 15 compounds (18 carbon signals) comprising carbons atoms bearing 1–4 chlorine atoms leads to an estimated error per chlorine atom of about 10–12 ppm. Specifically, linear regression of the data obtained at B3LYP/cc-pVDZ leads to y = −3.77 + 13.11x, with R = 0.982. Ultimately, experiment and theory converge for sym-pentachlorocorannulene and decachlorocorannulene, the former by correction of the theory, the latter by collecting the proper experimental data. Contribution to the Mark S. Gordon 65th Birthday Festschrift Issue.     

Magnetic susceptibility-induced alignment of proteins in reverse micelles

Proteins encapsulated within the aqueous core of reverse micelles are found to partially align in a magnetic field. The degree of alignment is sufficient to result in sizable residual 15N-1H dipolar couplings that can be easily measured. It is found that the magnetic susceptibility of the reverse micelle particle is not dominated by the encapsulated protein. The residual dipolar couplings are found to be structurally meaningful.