Editing of Chemical Exchange-Relayed NOEs in NMR Experiments for the Observation of Protein–Water Interactions

An experimental approach for the editing of exchange-relayed NOEs in water-selective NOE experiments is presented. The proposed pulse sequence is based on the application during the NOE mixing time of continuous wave irradiation, which saturates resonances of relaying labile protons in slow chemical exchange with water. The technique can efficiently reduce the contributions of exchange-relayed NOE peaks that often crowd the water-selective NOE spectra and hide direct intermolecular NOEs between water and protein protons. The present approach opens new opportunities for the characterization of hydration by NMR, even in the proximity of polar labile groups.     

Backbone-only restraints for fast determination of the protein fold: the role of paramagnetism-based restraints. Cytochrome b562 as an example

CH(alpha) residual dipolar couplings (Deltardc's) were measured for the oxidized cytochrome b562 from Escherichia coli as a result of its partial self-orientation in high magnetic fields due to the anisotropy of the overall magnetic susceptibility tensor. Both the low spin iron (III) heme and the four-helix bundle fold contribute to the magnetic anisotropy tensor. CH(alpha) Deltardc's, which span a larger range than the analogous NH values (already available in the literature) sample large space variations at variance with NH Deltardc's, which are largely isooriented within alpha helices. The whole structure is now significantly refined with the chemical shift index and CH(alpha) Deltardc's. The latter are particularly useful also in defining the molecular magnetic anisotropy parameters. It is shown here that the backbone folding can be conveniently and accurately determined using backbone restraints only, which include NOEs, hydrogen bonds, residual dipolar couplings, pseudocontact shifts, and chemical shift index. All these restraints are easily and quickly determined from the backbone assignment. The calculated backbone structure is comparable to that obtained by using also side chain restraint. Furthermore, the structure obtained with backbone only restraints is, in its whole, very similar to that obtained with the complete set of restraints. The paramagnetism based restraints are shown to be absolutely relevant, especially for Deltardc's.     

The fumarate sensor DcuS: progress in rapid protein fold elucidation by combining protein structure prediction methods with NMR spectroscopy

We illustrate how moderate resolution protein structures can be rapidly obtained by interlinking computational prediction methodologies with un- or partially assigned NMR data. To facilitate the application of our recently described method of ranking and subsequent refining alternative structural models using unassigned NMR data [Proc. Natl. Acad. Sci. USA 100 (2003) 15404] for such "structural genomics"-type experiments it is combined with protein models from several prediction techniques, enhanced to utilize partial assignments, and applied on a protein with an unknown structure and fold. From the original NMR spectra obtained for the 140 residue fumarate sensor DcuS, 1100 1H, 13C, and 15N chemical shift signals, 3000 1H-1H NOESY cross peak intensities, and 209 backbone residual dipolar couplings were extracted and used to rank models produced by de novo structure prediction and comparative modeling methods. The ranking proceeds in two steps: first, an optimal assignment of the NMR peaks to atoms is found for each model independently, and second, the models are ranked based on the consistency between the NMR data and the model assuming these optimal assignments. The low-resolution model selected using this ranking procedure had the correct overall fold and a global backbone RMSD of 6.0 angstrom, and was subsequently refined to 3.7 angstrom RMSD. With the incorporation of a small number of NOE and residual dipolar coupling constraints available very early in the traditional spectral assignment process, a model with an RMSD of 2.8 angstrom could rapidly be built. The ability to generate moderate resolution models within days of NMR data collection should facilitate large scale NMR structure determination efforts.     

Spin-lattice relaxation times and nuclear overhauser enhancement effect for P metabolites in model solutions at two frequencies: Implications for in vivo spectroscopy

The relaxation time T₁ values and nuclear Overhauser enhancement factor for ³¹P signal were determined in model solutions of metabolites ATP, PCr and Pi, and AMP at two frequencies and in H₂O and ²H₂O solutions. The data were analyzed to resolve the contribution of different relaxation mechanisms. A knowledge of NOE is important in the light of recent applications of double resonance methods to enhance the sensitivity of in vivo ³¹P spectroscopy. The results show that chemical shift anisotropy is the dominant mechanism for ³¹P in ATP at the high field, whereas the dipolar interaction mechanism is the main feature for the ³¹P relaxation of PCr and Pi. The dipolar mechanism responsible for NOE originates from interactions of solvent water with ³¹P moiety. Implications for in vivo spectroscopy are indicated.     

X-filtering for a range of coupling constants: application to the detection of intermolecular NOEs

A new method for heteronuclear X-filtering is presented, which relies on repetitive applications of 90 degrees (1H)-tau(1/41J(HC))-180 degrees (1H,13C)-tau(1/41J(HC))-90 degrees (1H,13C)-PFG building blocks employing gradient-mediated suppression of magnetization built up for directly heteronuclear coupled protons. Thereby, a range of heteronuclear coupling constants can be suppressed by varying the delays of scalar coupling evolution both within and between individual transients. To achieve efficient destruction of 13C-coupled protons in macromolecular systems, the scalar coupling evolution delays were optimized using simulated annealing by including transverse relaxation effects. With a combination of regular hard pulses, delays and pulsed field gradients only, this method yields sufficient X-filtering to allow the observation of intermolecular nuclear overhauser effects in a molecular complex consisting of a 13C, 15N double-labeled, and an unlabeled protein. This is achieved by exciting magnetization of 12C- and 14N-bound protons and detecting 13C-bound 1H magnetization in a 3D 13C-filtered, 13C-edited NOESY-HSQC experiment. The method is tested on the 18 kDa homodimeric bacterial antidote ParD.     

LR-CAHSQC: an application of a Carr-Purcell-Meiboom-Gill-type sequence to heteronuclear multiple bond correlation spectroscopy

A new pulse sequence, long-range CPMG-adjusted heteronuclear single quantum coherence (LR-CAHSQC), is proposed for the determination of long-range JCH coupling constants from a long-range 1H-13C correlation experiment. The long-range heteronuclear coupling constants can be directly extracted from COSY-type antiphase peak patterns. The current approach utilizes CPMG-sequences for polarization transfer, and thus avoids the evolution of homonuclear JHH couplings, which normally may introduce abnormalities into the cross peak pattern. The differences between LR-CAHSQC and normal LR-HSQC are discussed.     

Thomas-Fermi model for an atom in a very strong magnetic field: thermal effects

Thermal effects on the Thomas-Fermi model for atoms in a very strong magnetic field are considered. The binding energy of the atom is calculated with the thermal effects taken into account.     

Sensitivity to local dipole fields in the CRAZED experiment: an approach to bright spot MRI

Local dipole fields such as those created by small iron-oxide particles are used to produce regions of low intensity (dark contrast) in many molecular magnetic resonance imaging applications. We have investigated, with computer simulations and experiments at 17.6 T, how the COSY revamped with asymmetric z-gradient echo detection (CRAZED) experiment that selects intermolecular double-quantum coherences can also be used to visualize such local dipole fields. Application of the coherence-selection gradient pulses parallel to the main magnetic field produced similar, dark contrast as conventional gradient echo imaging. Application of the gradient along the magic angle leads to total loss of signal intensity in homogeneous samples. In the presence of local dipole fields, the contrast was inverted and bright signals from the dipoles were observed over a very low background. Both simulations and experiments showed that the signal strongly decreased when a phase-cycle suppressing single-quantum coherences was employed. Therefore, we conclude that most of the signal comes from directly refocused magnetization or intermolecular single-quantum coherences. Finally, we demonstrate that bright contrast from local dipole fields can also be obtained, when the pair of coherence-selection gradient pulses is deliberately mismatched. Both methods allowed visualization of local dipole fields in phantoms in experimental times of about 3 min.     

Liquid refractometry: an approach for a continuous measurement

A method is described for obtaining an accurate real-time measurement of the refractive index of liquids. It is based on a modification of the Pulfrich refractometer. The optical arrangement is suitable for in-line industrial applications where a continuous measurement is required. Some experimental results are presented which confirm the feasibility of the method.     

An improved method for suppressing protein background in PFG NMR experiments to determine ligand diffusion coefficients in the presence of receptor

In NMR diffusion experiments to study ligand-protein binding equilibria, the spectral background due to broad protein resonances can contribute significantly to the measured ligand signal intensity resulting in erroneous binding affinities. One method to suppress the protein spectral background involves coupling a CPMG pulse train before or after the BPPSTE pulse sequence to allow for differential T(2) relaxation of the broad protein resonances. Here, we present an improved method, the Gradient Phase Encoded Spin-lock (GraPES) experiment that integrates the relaxation filter into the diffusion period. Compared with sequential CPMG-BPPSTE pulse sequences, GraPES offers effective suppression of the protein background with improved signal-to-noise ratios and shorter experiment times.     

Plasmon imaging: an efficient TEM-based method for locating noble metal particles dispersed on oxide catalysts at very low densities

We have used transmission electron microscopy to study catalysts comprising nanoparticulate gold dispersed on a highly porous nanoparticulate TiO₂ (anatase) support. The similarity of the morphology of the two phases, and the low number density of gold particles (1 in 65,000) makes this challenging. Diffraction contrast imaging could not differentiate the two phases, since TiO₂ oriented at strong Bragg conditions, produced similar contrast to the Bragg/mass-thickness contrast of the gold. Mass-thickness contrast imaging allowed gold to be differentiated from TiO₂ only in the thinnest regions, where the mass-thickness of TiO₂ was low. Plasmon imaging, using an energy loss of 24 eV and an energy window width of 5 eV, was very effective at locating gold. Both the TiO₂ and impregnating resin produced a strong plasmon signal, while the much weaker signal from the gold made it appear dark. This permitted the gold particles to be readily located, irrespective of whether they were located in the thin or thick regions of the TiO₂ support.     

Fractional volume integration in two-dimensional NMR spectra: CAKE, a Monte Carlo approach

Quantitative information from multi-dimensional NMR experiments can be obtained by peak volume integration. The standard procedure (selection of a region around the chosen peak and addition of all values) is often biased by poor peak definition because of peak overlap. Here we describe a simple method, called CAKE, for volume integration of (partially) overlapping peaks. Assuming the axial symmetry of two-dimensional NMR peaks, as it occurs in NOESY and TOCSY when Lorentz-Gauss transformation of the signals is carried out, CAKE estimates the peak volume by multiplying a volume fraction by a factor R. It represents a proportionality ratio between the total and the fractional volume, which is identified as a slice in an exposed region of the overlapping peaks. The volume fraction is obtained via Monte Carlo Hit-or-Miss technique, which proved to be the most efficient because of the small region and the limited number of points within the selected area. Tests on simulated and experimental peaks, with different degrees of overlap and signal-to-noise ratios, show that CAKE results in improved volume estimates. A main advantage of CAKE is that the volume fraction can be flexibly chosen so as to minimize the effect of overlap, frequently observed in two-dimensional spectra.     

The rapid determination of fat and protein content in fresh raw milk using the laser light scattering technology

The aim was to develop a simple and rapid method for determination of fat and protein content in milk. Based on the laser light scattering theory, the ratio of the scattered light (at 90±0.05° scattering angles) intensity to the transmitted light intensity, which is called scattered–transmitted-ratio method, is adopted as the optical parameter representing the milk fat content and the protein content. In this way, the influence of the fluctuation of the power of the light source is eliminated and the accuracy of determination is improved accordingly. The system we use is real-time and can satisfy the challenging requirements of dairy farming. Results of this study indicate the feasibility of using this technology for fresh milk fat and protein analysis. The fat contents and protein contents of 50 milk samples determined by this method were consistent with the values obtained by the reference methods based on Rose-Gottlieb method and Kjeldahl determination of N method. In this paper, the operating principle of the instrument is introduced and the influence of the environmental conditions, such as the homogenization pressure and homogenization temperature, etc. on the result of the test is analyzed. Through data analysis, the concrete schemes for testing the fat using the curve fitting and testing the protein using the surface fitting technique are determined. Finally, the difference from the reference values of the test is discussed.     

自旋回波的简易观测方法及共振弛豫分析

在脉冲核磁共振实验中,通常采用自旋回波法测量共振弛豫时间,但模拟示波器观测难以获得准确的实验数据.通过对计算机标准配置资源声卡的性能检测及标定,使其达到物理实验测量数据定量分析的教学要求,同时利用免费的简易程序实现多通道数字信号采集功能并用于观测记录脉冲核磁共振信号.配合实验操作技术改进,既准确地测量了横向弛豫时间,又展现了符合物理实验教学的计算机应用方法.     

A new method for generation of quasi-periodic structures withn fold axes: Application to five and seven folds

A new geometrical method for generating aperiodic lattices forn-fold non-crystallographic axes is described. The method is based on the self-similarity principle. It makes use of the principles of gnomons to divide the basic triangle of a regular polygon of 2n sides to appropriate isosceles triangles and to generate a minimum set of rhombi required to fill that polygon. The method is applicable to anyn-fold noncrystallographic axis. It is first shown how these regular polygons can be obtained and how these can be used to generate aperiodic structures. In particular, the application of this method to the cases of five-fold and seven-fold axes is discussed. The present method indicates that the recursion rule used by others earlier is a restricted one and that several aperiodic lattices with five fold symmetry could be generated. It is also shown how a limited array of approximately square cells with large dimensions could be detected in a quasi lattice and these are compared with the unit cell dimensions of MnAl₆ suggested by Pauling. In addition, the recursion rule for sub-dividing the three basic rhombi of seven-fold structure was obtained and the aperiodic lattice thus generated is also shown. Based on the lecture by the author “Quasi crystals: Is Linus Pauling right” and delivered on 16 December 1985 and arranged by the Departments of Physics, Metallurgy, Materials Research Laboratory, and Instrumentation Services Unit, Indian Institute of Science, Bangalore.     

Yukawa alignment in a multi Higgs doublet model: An effective approach

In the two Higgs doublet model, natural flavour conservation can be achieved through the use of a discrete Z₂$Z_2$ symmetry. A less restrictive condition is the requirement of alignment in the Yukawa sector. So far, alignment has been an ansatz, not rooted in a specific model. In this Letter we present a model for alignment, which starts with 2+N$2+N$ Higgs doublets, with natural flavour conservation imposed by a discrete symmetry. Only two of these scalars couple to the fermions, the other N scalars are in a hidden sector. Assuming that the two scalar doublets coupled to fermions are heavy, their decoupling leads to an effective Yukawa interaction. The latter connects the fermions and the scalars of the hidden sector, and exhibits the same Yukawa coupling matrix for each of the N scalars.     

Free-swarm method: a new approach for measuring electron drift velocity in a gas in an electric field

Summary A new method is presented for measuring electron drift velocityW in a gas in a constant and homogeneous electric field. The technique does not require any correction for electrode and diffusion effects and can give ?instantaneous? values ofW (that is, values measured in a time interval that is limited with respect to the total drift time). We have tested this methodvs. standard methods with good results; it may be useful in the presence of non-stationary behaviour ofW.     

A thin-film transistor with a very thin a-Si:H layer and its application for an LC panel

One of the disadvantages of applying an a-Si:H thin-film transistor (TFT) to an active matrix-addressed liquid crystal (LC) panel is that a TFT with an a-Si:H has a very large photo-leakage current because of the high photo-conductivity of an a-Si:H itself.We have tried decreasing the photo-leakage current by varying the thickness of an a-Si:H layer (L) in TFTs and investigated the characteristics of TFTs, mainly drain voltage versus drain current containing photo-leakage current (I _ph).As a result, it is shown that lnI _ph is proportional to InL, and its gradient is 1.5–2.0. We assume that the thinner an a-Si:H layer is, the more effective the recombination of carriers at the interface states is forI _ph.We have applied TFT with a very thin a-Si:H layer (30nm) to a full-color active matrix-addressed LC panel for a moving picture display and realized a display of good quality under illuminated condition of 5×10⁴lx without a shading layer in it.     

Ultrasound promoted aminolysis of epoxides in aqueous media: a rapid procedure with no pH adjustment for additive-free synthesis of beta-aminoalcohols

An efficient and environmentally friendly procedure promoted by ultrasound irradiation is developed for stereoselective ring opening of various epoxides with aromatic and aliphatic amines under aqueous conditions in the presence of no catalyst or additive. Chemoselectivity of the protocol is shown by competition of piperidine and aniline to react with different epoxides resulting in exclusive formation of the respective products of piperidine.