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Structural studies of polytopic membrane proteins are often hampered by the vagaries of these proteins in membrane mimetic environments and by the difficulties in handling them with conventional techniques. Designing and creating water-soluble analogues with preserved native structures offer an attractive alternative. We report here solution NMR studies of WSK3, a water-soluble analogue of the potassium channel KcsA. The WSK3 NMR structure (PDB ID code 2K1E) resembles the KcsA crystal structures, validating the approach. By more stringent comparison criteria, however, the introduction of several charged residues aimed at improving water solubility seems to have led to the possible formations of a few salt bridges and hydrogen bonds not present in the native structure, resulting in slight differences in the structure of WSK3 relative to KcsA. NMR dynamics measurements show that WSK3 is highly flexible in the absence of a lipid environment. Reduced spectral density mapping and model-free analyses reveal dynamic characteristics consistent with an isotropically tumbling tetramer experiencing slow (nanosecond) motions with unusually low local ordering. An altered hydrogen-bond network near the selectivity filter and the pore helix, and the intrinsically dynamic nature of the selectivity filter, support the notion that this region is crucial for slow inactivation. Our results have implications not only for the design of water-soluble analogues of membrane proteins but also for our understanding of the basic determinants of intrinsic protein structure and dynamics. membrane protein | protein design | potassium channels | slow inactivation

Field map processing and NMT lineshape analysis methods are described for using in the field improvement techniques specifically applied to 700 MHz LTS/HTS NMR magnet. A high-resolution GHz-class low temperature superconductor (LTS)/high temperature superconductor (HTS) NMR magnet is obtained by understanding the mechanisms of screening current induced field (SCF) in LTS/HTS NMR magnets.

A high-field NMR magnet including high-temperature superconductor (HTS) coils is operated in the driven-mode and the magnetic field drift of a 14 Tesla NMR magnet is evaluated in the driven mode. The shielding current in the damper coil has cancelled out the field fluctuation, thus indicating that the damper coil has worked effectively.

High-field NMR magnet including high temperature superconductor (HTS) coils are operated in the driven-mode and the magnetic field stability is evaluated in the driven mode by modifying a 14 Tesla (600 MHz) vertical NMR magnet. The residual fluctuation in the driven-mode is large when compared to the persistent-mode and the fluctuations are not preferred for NMR measurements.

The chaperonin GroEL binds non-native polypeptides in an open ring via hydrophobic contacts and then, after ATP and GroES binding to the same ring as polypeptide, mediates productive folding in the now hydrophilic, encapsulated cis chamber. The nature of the folding reaction in the cis cavity remains poorly understood. In particular, it is unclear whether polypeptides take the same route to the native state in this cavity as they do when folding spontaneously free in solution. Here, we have addressed this question by using NMR measurements of the time course of acquisition of amide proton exchange protection of human dihydrofolate reductase (DHFR) during folding in the presence of methotrexate and ATP either free in solution or inside the stable cavity formed between a single ring variant of GroEL, SR1, and GroES. Recovery of DHFR refolded by the SR1/GroES-mediated reaction is 2-fold higher than in the spontaneous reaction. Nevertheless, DHFR folding was found to proceed by the same trajectories inside the cis folding chamber and free in solution. These observations are consistent with the description of the chaperonin chamber as an 'Anfinsen cage' where polypeptide folding is determined solely by the amino acid sequence, as it is in solution. However, if misfolding occurs in the confinement of the chaperonin cavity, the polypeptide chain cannot undergo aggregation but rather finds its way back to a productive pathway in a manner that cannot be accomplished in solution, resulting in the observed high overall recovery. chaperonin reaction | protein folding | GroEL-mediated folding | NMR | solvent protection

Critical fluctuations are investigated in lipid membranes near miscibility critical points in bilayers composed of dioleoylphosphatidylcholine, chain perdeuterated dipalmitoylphosphatidylcholine, and cholesterol. Phase boundaries are mapped over the temperature range from 10[degrees]C to 60[degrees]C by deuterium NMR. Tie-lines and three-phase triangles are evaluated across two-phase and three-phase regions, respectively. In addition, a line of miscibility critical points is identified. NMR resonances are broadened in the vicinity of critical points, and broadening is attributed to increased transverse relaxation rates arising from modulation of chain order with correlation times on a microsecond time scale. We conclude that spectral broadening arises from composition fluctuations in the membrane plane with dimensions of lipid rafts | liquid-immiscibility | NMR relaxation | phase diagram | critical behavior

We propose to use infrared coherent two-dimensional correlation spectroscopy (2DCS) to characterize the fibril structure of Ab42, the dominant composition of Ab deposit, which is crucial for investigating its toxicity and aggregation mechanism. By optimizing the pulse polarization configurations with a genetic algorithm combined with sensitivity analysis, we obtained signals with well resolved cross-peak features attributed to the couplings within and between different structural motifs. These signals may provide new constraints for refining of the currently available NMR structure. Two-dimensional correlation spectroscopy also can differentiate the turn structure of Ab42 and other Ab derivatives. correlation spectroscopy | four wave mixing

Assessment determines that nuclear magnetic resonance spectroscopy shows limitations in quantifying in vivo whole-body carbohydrate metabolism. Limitations stem from difficulties associated with conversion of a spectroscopic signal into a reliable and interpretable biochemical concentration, and then into a flux rate.

Abstract: From the (automorphic) nature of nuclear permutational NMR spin symmetry established in the 1980s, the SU(2)\ \ S.sub.n tensorial sets exhibit simple reducibility (SR) over their pattern algebraicabased Liouvillian carrier space [F.P. Temme, Physica A 166 (1990) 676 198 (1993) 245 Chem. Phys. 238 (1998) 245] for NMR. This underlies the quantum physical role of the S.sub.n group and its fundamental set of system invariants (SIs), i.e. beyond the Cartesian SIs of [P.L. Corio, J. Magn. Reson. 134 (1998) 131]. Here we examine the outerarank dualagroup tensorial subsets (over a \ [[tilde over (term)][lambda] ]\ (S.sub.n ) field) as standardised maximal forms for all [lambda] | - n 1pt : ([lambda] Article History: Registration Date: 12/10/2004

The National High Magnetic Field Laboratory is tasked with developing applications of high-field magnets. The laboratory, which has facilities located at Florida State University in Tallahasee, the University of Florida in Gainesville and the Los Alamos National Laboratory in New Mexico, is also tasked with finding applications of superconducting magnets such as nuclear magnetic resonance, electron spin resonance and ion cyclotron resonance in physics and analytical chemistry.

A novel method to the empirical study of vicinal coupling constants and NOE effects is presented. It employs the maximum-entropy probability density distribution, which can successfully replicate complex Gaussian distributions. The technique can be employed in the study of side-chain conformations in carbohydrates such as exocyclic, amino and hydroxyl groups. It can be extended to the analysis of conformations of several glycosidic linkages.

A study was done on the biosynthesis of C-terminal alpha-amidated peptides (alpah-AE) from C-terminal glycine-extended precursors using bifunctional type A rat alpha-AE. It was shown that two moles of ascorbate undergo oxidation during amidation to produce two moles of semidehydroascorbate. Futhermore, an 18O-13C nuclear magnetic resonance isotopic shift shows that O2 is the source of the alpha-carbonyl oxygen of pyruvate generated during the amidation of dansyl-Tyr-Val-D-Ala.

We present a novel test tube magnet (TTM) for use in portable nuclear magnetic resonance (NMR) devices. The TTM consists of a dipole cylinder magnet and a hemisphere magnet. The hemisphere magnet is attached to one end of the dipole cylinder magnet, so that the whole magnet is shaped like a test tube. TTM generates a wide-diameter spherical volume (DSV) with a high magnetic field homogeneity, which makes the device suitable for portable NMR. We simulated the configuration with three-dimensional finite-element methods. Flux density elements are effectively corrected in the homogeneous magnetic field region. The homogeneity increases, the magnet volumes and mass relatively decrease, and the construction is fairly simple. Overall, the new design has improved characteristics for portable NMR. Index Terms--Field compensation, Halbach arrays, portable NMR, test tube magnet (TTM).

A newly developed NMR titration approach is employed to explain the various position specific secondary isotope effects observed on the basicities of the various deuterated pyridine isotopologues. The basicity is shown to significantly enhance with the deuteration process.

A study was conducted to examine the complete covalent structures of two purified closely related antifungal compounds, which were observed by the experiments of TOCSY, NOESY, ROESY, [super 13]C HSQC 2D NMR, and ESI-MS and GC. Findings suggest that the antifungal compound interrupts normal membrane morphology.

A study was conducted to measure water-soluble inositol 1,2-(cyclic) phosphate (cIP) adhering to phosphatidylinositol-specific phospholipase C spin-labeled at H82C using [super 31]P NMR from 0.005 to 11.7 T.

The isomers of cinnamoyl [alpha]-CDs (2-CiO-[alpha]-CD) have organized themselves to give different types of supramolecular complexes in aqueous solution, where 2-CiO-[alpha]-CD is shown to form a double-threaded dimer. The studies have shown that CDs have recognized a guest molecule and the substantial position of a guest molecule on CD, indicating that CDs have social self-sorting capability.

Less dynamic cross-link models such as (R,S,S,R)-BipPt(d(TG*G*)) and (R,S,S,R)-BipPt(d(pG*G*TTT)), the first examples of an HH1 conformer with R canting for single-strand (ss) oligos longer than d(GpG) (Bip = 2,2'-bipiperidine) is used for defining the ss features. The studies have shown that the 5'-X residue in duplex adducts has changed its position from that in B form DNA to avoid steric clashes with the 5'-G* and the carrier ligand and also to form a Watson-Crick base pair.

The article studies the crystal structure, electric field gradient (EFG), and electronic charge densities in Re[B.sub.2]. Results conclude that the principal axis of the most negative EFG-tensor component [V.sub.zz] is parallel to the c-axis of the crystal.

Five new compounds are prepared for using as acid amplifiers in EUV photoresists and decomposition rates are studied by using [super 19]F NMR in the presence and absence of 1.2 equiv of tri-tert-butylpyridine. Preliminary lithographic evaluation has shown that cis-1-methyl-2-(4-(trifluoromethyl)phenylsulfonyloxy)cyclohexyl acetate has improved the resolution, line edge roughness (LER) and sensitivity of an EUV photoresist.

Solid state nuclear magnetic resonance is used for examining the carbon nanotubes functionalized with sulfonated polyether-ether-ketone. The results are interpreted at the molecular level in terms of magnetic ring currents originating from the surface of the graphitized wall of the nanotubes, where these features are used to track the structural modifications.

The feasibility of elucidating the bound conformation of a periplasmic binding protein, the glutamine-binding protein (GlnBP), in solution is demonstrated by using paramagnetic relaxation enhancements (PREs) arising from a single paramagnetic group. The ligand-free conformation is used as a starting point for performing conjoined rigid-body/torsion-angle simulated annealing calculations by using backbone [super 1][H.sup.N]-PREs as a major source of distance information.

Accurate measurement of the very small residual chemical shift anisotropy (RCSA) from the difference in [super 15]N chemical shift under isotropic and weakly aligning liquid crystalline conditions is very sensitive to minute differences in sample conditions, such as pH or ionic strength. Fitting the RCSA data together with [super 15]N-[super 1]H dipole-CSA cross-correlated relaxation measurements to the refined structural model of t[RNA.sup.Val] has produced the magnitude, asymmetry and orientation of the [super 15]N CSA tensors.

Molecular dynamics simulations of NMR backbone relaxation order parameters are carried out to examine the polarization effect on the protein's local structure and dynamics for five benchmark proteins. The analysis of the structures and dynamic motions of individual residues have shown that the hyperflexibility of these local structures is related to the breaking or weakening of relevant hydrogen bonds.

An isotopically discriminated IDIS-residual dipolar coupling (RDC)-TROSY NMR experiment is proposed that has enabled the measurement of HN RDCs for two proteins simultaneously and independently, but in the same sample, while they are part of the same complex. The method has ensured that RDCs measured for both proteins have related to exactly the same alignment tensor, allowing accurate measurement of the relative angle between the two proteins.

The Selective Excitation-Inversion (SEI) Antagonist Induced Dissociation Assay (AIDA) method is described that has used tryptophan-bearing proteins. The acquisition time of the AIDA experiment is reduced by an order of magnitude relative to the corresponding 1D AIDA using hard pulses by selectively exciting only the proton NMR signals of the [super N][H.sub.[epsilon]] indole side chains of tryptophans.

A solid-state NMR and site-directed spin labeling/EPR with a synthetic peptide are used to study the influence of membrane environment on human cannabinoid 1 (h[CB.sub.1]) receptor transmembrane helix (TMH) conformational dynamics. The data obtained indicated that h[CB.sub.1](T377-E416) backbone dynamics as well as spin-label rotameric freedom are sensitive to and altered by the phospholipid bilayer environment of peptide, which exerted a dynamic influence on the conformation of a TMH critical to signal transmission by the h[CB.sub.1] receptor.

Nuclear magnetic resonance and synchrotron radiation circular dichroism (SRCD) spectroscopy were employed to demonstrate the differances in conformation changes observed for N-acetyl-enriched heparin polysaccharide in the presence of sodium or copper(II) cations. The cations associated with an N-acetyl-enriched heparin polysaccharide on change from sodium to copper(II), converted it from supporting signaling through the fibroblast growth factor receptor (FGF-1-FGFR1c) tyrosine kinase signaling system to being inhibitory in a cell-based BaF3 assay.

The nature of the substrate-catalyst complex and its dynamic properties are examined by NMR methods and with the help of a model compound lacking the epoxide function. The kinetic parameters of the cyclization reaction are studied, a catalytic cycle is proposed and diverse inhibition mechanisms are identified that parallel those encountered in enzymology.

Circular dichroism, fluorescence polarization and NMR are used for showing the presence of an additional binding site located between those for two c-Myc-Max inhibitors, 10058-F4 and 10074-G5. The results have shown the existence of peptide regions prone to small-molecule binding within intrinsically disordered (ID) domains.

A kinetically controlled approach is described for preparing thiolate-capped 20-atom gold clusters. The [Au.sub.20][(SC[H.sub.2]C[H.sub.2]Ph).sub.16] clusters have displayed a multiple-band optical absorption spectrum and the optical energy gap is determined to be 2.15 eV.

The enzyme triosephosphate isomerase (TIM) was used as a model system to understand the relationship between protein sequence, structure, and biological function and the direct correlation between sequence of the active site loop (loop 6) in TIM and a conserved motif in loop 7. The result data provide insight into the significance of loop 6 and loop 7 to provide the proper chemical context for the enzymatic reaction and the singnifican role of interactions in modulation of the chemical dynamics near the active site.

The liquid samples are polarized in situ using a double-resonance structure, which allowed simultaneous excitation of NMR and EPR transitions and has achieved significant dynamic nuclear polarization (DNP) enhancements at very low incident microwave power of only 45 mW. The results have shown the important step toward the application of DNP to high-resolution NMR, increasing the sensitivity on biomolecules with small sample volumes and at physiologically low concentrations.