Pressure-assisted cold denaturation of hen egg white lysozyme: the influence of co-solvents probed by hydrogen exchange nuclear magnetic resonance

COSY proton nuclear magnetic resonance was used to measure the exchange rates of amide protons of hen egg white lysozyme (HEWL) in the pressure-assisted cold-denatured state and in the heat-denatured state. After dissolving lysozyme in deuterium oxide buffer, labile protons exchange for deuterons in such a way that exposed protons are substituted rapidly, whereas "protected" protons within structured parts of the protein are substituted slowly. The exchange rates k obs were determined for HEWL under heat treatment (80ºC) and under high pressure conditions at low temperature (3.75 kbar, -13ºC). Moreover, the influence of co-solvents (sorbitol, urea) on the exchange rate was examined under pressure-assisted cold denaturation conditions, and the corresponding protection factors, P, were determined. The exchange kinetics upon heat treatment was found to be a two-step process with initial slow exchange followed by a fast one, showing residual protection in the slow-exchange state and P-factors in the random-coil-like range for the final temperature-denatured state. Addition of sorbitol (500 mM) led to an increase of P-factors for the pressure-assisted cold denatured state, but not for the heat-denatured state. The presence of 2 M urea resulted in a drastic decrease of the P-factors of the pressure-assisted cold denatured state. For both types of co-solvents, the effect they exert appears to be cooperative, i.e., no particular regions within the protein can be identified with significantly diverse changes of P-factors.

Polytypism and Silicon carbide : a solid state nuclear magnetic resonance study

A survey of predominantly industrial silicon carbide has been carried out using Magic Angle Spinning nuclear magnetic resonance (MAS nmr); a solid state technique. Three silicon carbide polytypes were studied; 3C, 6H, and 15R. The 13C and 29 Si MAS nmr spectra of the bulk SiC sample was identified on the basis of silicon (carbon) site type in the d iff ere n t pol Y t Y pes • Out to 5.00 A fro mac en t r a lsi 1 i con (0 r carbon) atom four types of sites were characterized using symmetry based calculations. This method of polytype analysis was also considered, in the prelminary stages, for applications with other polytypic material; CdBr 2 , CdI 2 , and PbI 2 " In an attempt to understand the minor components of silicon carbide, such as its surface, some samples were hydrofluoric acid washed and heated to extreme temperatures. Basically, an HF removable species which absorbs at -110 ppm (Si0 2 ) in the 29 Si MAS nmr spectrum is found in silicon carbide after heating. Other unidentified peaks observed at short recycle delays in some 29 Si MAS nmr spectra are considered to be impurities that may be within the lattice. These components comprise less than 5% of the observable silicon. A Tl study was carried out for 29 Si nuclei in a 3C ii polytype sample, using the Driven Equilibrium Single-Pulse Observation of T1 (DESPOT) technique. It appears as though there are a number of nuclei that have the same chemical shift but different T1 relaxation times. The T1 values range from 30 seconds to 11 minutes. Caution has to be kept when interpreting these results because this is the first time that DESPOT has been used for solid samples and it is not likely in full working order. MAS nmr indicates that the 13C and 29 Si ~sotropic chemical shifts of silicon carbide appear to have a reciprocal type of relationship_ Single crystal nmr analysis of a 6H sample is accordance with this finding when only the resultant isotropic shift is considered. However, single crystal nmr also shows that the actual response of the silicon and carbon nuclear environment to the applied magnetic field at various angles is not at all reciprocal. Such results show that much more single crystal nmr work is required to determine the actual behavior of the local magnetic environment of the SiC nuclei.

Nuclear magnetic resonance studies of boron trihalide complexes of 1,1-BIS (dimethylamino) ethylene and related bases

Boron tribalide complexes of 1,1-bis(dimethylamino)ethylene (DME) , t etramethylurea (TMU), tetramethylguanidine (TMG) , and pentamethylguanidine (PMG) and also mixed boron t r ihalide adducts of DME have been investigated by 1H and 19F NMR spectroscopy. Both nitrogen and the C-Q-H carbon of DME are possible donor a toms to boron trihal ides but complexation has been found to occur only at carbon of DME. The initial adduct acts as a Bronsted acid and gives up a proton to free DME in solut ion. A side reaction in the DME-BF, system gives rise to trace amounts of a complex aSSigned as (DME)2BF2+. (DME)2BF2+ is produced in much larger quantities in t he DME-BF3-BC13 and DME-BF,-BBr, systems by reaction of free DME with DME:BF2X (X = Cl, Br). Restricted r otation about the C-N bonds of TMUlBC13 and n1U:BBr3 has been observed at low temperatures. This complements previous work in this system and confirms oxygen donation of TMU to boron trihalides . Restricted rotation at low temperatures also has been observed in DMEboron trihalide systems

Effects of hypothermia on brain glucose metabolism in acute liver failure: a H/C-nuclear magnetic resonance study.

Mild hypothermia has a protective effect on brain edema and encephalopathy in both experimental and human acute liver failure. The goals of the present study were to examine the effects of mild hypothermia (35°C) on brain metabolic pathways using combined 1H and 13C-Nuclear Magnetic Resonance (NMR) spectroscopy, a technique which allows the study not only of metabolite concentrations but also their de novo synthesis via cell-specific pathways in the brain. :1H and 13C NMR spectroscopy using [1-13C] glucose was performed on extracts of frontal cortex obtained from groups of rats with acute liver failure induced by hepatic devascularization whose body temperature was maintained either at 37°C (normothermic) or 35°C (hypothermic), and appropriate sham-operated controls. At coma stages of encephalopathy in the normothermic acute liver failure animals, glutamine concentrations in frontal cortex increased 3.5-fold compared to sham-operated controls (P < 0.001). Comparable increases of brain glutamine were observed in hypothermic animals despite the absence of severe encephalopathy (coma). Brain glutamate and aspartate concentrations were respectively decreased to 60.9% ± 7.7% and 42.2% ± 5.9% (P < 0.01) in normothermic animals with acute liver failure compared to control and were restored to normal values by mild hypothermia. Concentrations of lactate and alanine in frontal cortex were increased to 169.2% ± 15.6% and 267.3% ± 34.0% (P < 0.01) respectively in normothermic rats compared to controls. Furthermore, de novo synthesis of lactate and alanine increased to 446.5% ± 48.7% and 707.9% ± 65.7% (P < 0.001), of control respectively, resulting in increased fractional 13C-enrichments in these cytosolic metabolites. Again, these changes of lactate and alanine concentrations were prevented by mild hypothermia. Mild hypothermia (35°C) prevents the encephalopathy and brain edema resulting from hepatic devascularization, selectively normalizes lactate and alanine synthesis from glucose, and prevents the impairment of oxidative metabolism associated with this model of ALF, but has no significant effect on brain glutamine. These findings suggest that a deficit in brain glucose metabolism rather than glutamine accumulation is the major cause of the cerebral complications of acute liver failure.

Development of selected electro-active polymer-ceramic nanocomposites as pyroelectric thermal/infrared detector materials

The present work emphasises on the synthesis and characterization of electro-active polymer-ceramic nanocomposites which can be used for pyroelectric thermal/infrared detection applications. Two sets of samples belong to polymer-microcrystalline composites have also been investigated in the work. The polymers used in the work have been commercially available ones, but the nanoceramics have been synthesized following simple chemical routes and aqueous organic gel routes. After characterizing the nanoceramics for their structure by powder XRD, they have been dispersed in liquid polymer and sonicated for uniform dispersion. The viscous mixture so formed was cast in the form of films for experimentation. Samples with volume fraction of the ceramic phase varied from 0 to 0.25 have been prepared. Solution growth was followed to prepare microcrystalline samples for the polymer-microcrystalline composites. The physical properties that determine the pyroelectric sensitivity of a material are dielectric constant, dielectric loss, pyroelectric coefficient, thermal conductivity and specific heat capacity. These parameters have been determined for all the samples and compositions reported in this work.The pyroelectric figures of merit for all the samples were determined. The pyroelectric figures of merit that determine the pyroelectric sensitivity of a material are current sensitivity, voltage responsivity and detectivity. All these have been determined for each set of samples and reported in the thesis. In order to assess the flexibility and mouldability of the composites we have measured the Shore hardness of each of the composites by indentation technique and compared with the pyroelectric figures of merit. Some important factors considered during the material fabrication stages were maximum flexibility and maximum figures of merit for pyroelectric thermal/IR detection applications. In order to achieve these goals, all the samples are synthesized as composites of polymers and nano/microcrystalline particles and are prepared in the form of freestanding films. The selected polymer matrices and particle inclusions possess good pyroelectric coefficients, low thermal and dielectric properties, so that good pyroelectric figures of merit could be achieved. The salient features of the work include the particle size of the selected ceramic materials. Since they are in nanometer size it was possible to achieve high flexibility and moldability with high figures of merit for even low volume fractions of inclusions of the prepared nanocrystalline composites. In the case of microcrystalline TGS and DTGS, their composites in PU matrix protect them from fragility and humidity susceptibility and made them for environmental friendly applications.

Nuclear magnetic resonance structure shows that the severe acute respiratory syndrome coronavirus-unique domain contains a macrodomain fold

The nuclear magnetic resonance (NMR) structure of a central segment of the previously annotated severe acute respiratory syndrome (SARS)-unique domain (SUD-M, for "middle of the SARS-unique domain") in SARS coronavirus (SARS-CoV) nonstructural protein 3 (nsp3) has been determined. SUD-M(513-651) exhibits a macrodomain fold containing the nsp3 residues 528 to 648, and there is a flexibly extended N-terminal tail with the residues 513 to 527 and a C-terminal flexible tail of residues 649 to 651. As a follow-up to this initial result, we also solved the structure of a construct representing only the globular domain of residues 527 to 651 [SUD-M(527-651)]. NMR chemical shift perturbation experiments showed that SUD-M(527-651) binds single-stranded poly(A) and identified the contact area with this RNA on the protein surface, and electrophoretic mobility shift assays then confirmed that SUD-M has higher affinity for purine bases than for pyrimidine bases. In a further search for clues to the function, we found that SUD-M(527-651) has the closest three-dimensional structure homology with another domain of nsp3, the ADP-ribose-1 ''-phosphatase nsp3b, although the two proteins share only 5% sequence identity in the homologous sequence regions. SUD-M(527-651) also shows three-dimensional structure homology with several helicases and nucleoside triphosphate-binding proteins, but it does not contain the motifs of catalytic residues found in these structural homologues. The combined results from NMR screening of potential substrates and the structure-based homology studies now form a basis for more focused investigations on the role of the SARS-unique domain in viral infection.

Nuclear magnetic resonance structure of the nucleic acid-binding domain of severe acute respiratory syndrome coronavirus nonstructural protein 3

The nuclear magnetic resonance (NMR) structure of a globular domain of residues 1071 to 1178 within the previously annotated nucleic acid-binding region (NAB) of severe acute respiratory syndrome coronavirus nonstructural protein 3 (nsp3) has been determined, and N- and C-terminally adjoining polypeptide segments of 37 and 25 residues, respectively, have been shown to form flexibly extended linkers to the preceding globular domain and to the following, as yet uncharacterized domain. This extension of the structural coverage of nsp3 was obtained from NMR studies with an nsp3 construct comprising residues 1066 to 1181 [ nsp3(1066-1181)] and the constructs nsp3(1066-1203) and nsp3(1035-1181). A search of the protein structure database indicates that the globular domain of the NAB represents a new fold, with a parallel four-strand beta-sheet holding two alpha-helices of three and four turns that are oriented antiparallel to the beta-strands. Two antiparallel two-strand beta-sheets and two 3(10)-helices are anchored against the surface of this barrel-like molecular core. Chemical shift changes upon the addition of single-stranded RNAs (ssRNAs) identified a group of residues that form a positively charged patch on the protein surface as the binding site responsible for the previously reported affinity for nucleic acids. This binding site is similar to the ssRNA-binding site of the sterile alpha motif domain of the Saccharomyces cerevisiae Vts1p protein, although the two proteins do not share a common globular fold.

Nuclear magnetic resonance structure of the N-terminal domain of nonstructural protein 3 from the severe acute respiratory syndrome coronavirus

This paper describes the structure determination of nsp3a, the N-terminal domain of the severe acute respiratory syndrome coronavirus (SARS-CoV) nonstructural protein 3. nsp3a exhibits a ubiquitin-like globular fold of residues 1 to 112 and a flexibly extended glutamic acid-rich domain of residues 113 to 183. In addition to the four beta-strands and two alpha-helices that are common to ubiquitin-like folds, the globular domain of nsp3a contains two short helices representing a feature that has not previously been observed in these proteins. Nuclear magnetic resonance chemical shift perturbations showed that these unique structural elements are involved in interactions with single-stranded RNA. Structural similarities with proteins involved in various cell-signaling pathways indicate possible roles of nsp3a in viral infection and persistence.

Synthesis, structure and magnetic properties of mono- and di-nuclear nickel(II) thiocyanate complexes with tridentate N3 donor Schiff bases

The 1:1 condensation of N-methyl-1,3-diaminopropane and N,N-diethyl-1,2-diminoethane with 2-acetylpyridine, respectively at high dilution gives the tridentate mono-condensed Schiff bases N-methyl-N'-(1-pyridin-2-yl-ethylidene)-propane-1,3-diamine (L-1) and N,N-diethyl-N'-(1-pyridin-2-yl-ethylidene)-ethane-1,2-diamine (L-2). The tridentate ligands were allowed to react with methanol solutions of nickel(II) thiocyanate to prepare the complexes [Ni(L-1)(SCN)(2)(OH2) (1) and [{Ni(L-2)(SCN)}(2)] (2). Single crystal X-ray diffraction was used to confirm the structures of the complexes. The nickel(II) in complex 1 is bonded to three nitrogen donor atoms of the ligand L-1 in a mer orientation, together with two thiocyanates bonded through nitrogen and a water molecule, and it is the first Schiff base complex of nickel(II) containing both thiocyanate and coordinated water. The coordinated water initiates a hydrogen bonded 2D network. In complex 2, the nickel ion occupies a slightly distorted octahedral coordination sphere, being bonded to three nitrogen atoms from the ligand L-2, also in a mer orientation, and two thiocyanate anions through nitrogen. In contrast to 1, the sixth coordination site is occupied by a sulfur atom from a thiocyanate anion in an adjacent molecule, thus creating a centrosymmetric dimer. A variable temperature magnetic study of complex 2 indicates the simultaneous presence of zero-field splitting, weak intramolecular ferromagnetic coupling and intermolecular antiferromagnetic interactions between the nickel(II) centers.

Synthesis, crystal structure and magnetic properties of three unprecedented tri-nuclear and one very rare tetra-nuclear copper(II) Schiff-base complexes supported by mixed azido/phenoxo/nitrato or acetato bridges

Three novel mixed bridged trinuclear and one tetranuclear copper(II) complexes of tridentate NNO donor Schiff base ligands [Cu-3(L-1)(2)(mu(LI)-N-3)(2)(CH3OH)(2)(BF2)(2)] (1), [Cu-3(L-1)(2)(mu(LI)-NO3-I kappa O.2 kappa O')(2)] (2), [Cu-3(L-2)(2)(mu(LI)-N-3)(2)(mu-NOI-I kappa O 2 kappa O')(2)] (3) and [Cu-4(L-3)(2)(mu(LI)-N-3)(4)(mu-CH3COO-I kappa O 2 kappa O')(2)] (4) have been synthesized by reaction of the respective tridentate ligands (L-1 = 2[1-(2-dimethylamino-ethylimino)-ethyl]-phenol, L-2 = 2[1-(2-diethylamino-ethylimino)-ethyl]-phenol, L-3 = 2-[1-(2-dimethylamino-ethylimino)-methyl]-phenol) with the corresponding copper(II) salts in the presence of NaN3 The complexes are characterized by single-crystal X-ray diffraction analyses and variable-temperature magnetic measurements Complex 1 is composed of two terminal [Cu(L-1)(mu(LI)-N-3)] units connected by a central [Cu(BF4)(2)] unit through nitrogen atoms of end-on azido ligands and a phenoxo oxygen atom of the tridentate ligand The structures of 2 and 3 are very similar, the only difference is that the central unit is [Cu(NO1)(2)] and the nitrate group forms an additional mu-NO3-I kappa O 2 kappa O' bridge between the terminal and central copper atoms In complex 4, the central unit is a di-mu(L1)-N-3 bridged dicopper entity, [Cu-2(mu(L1)-N-3)(2)(CH3COO)(2)] that connects two terminal [Cu(L-3)(mu(L1)-N-3)] units through end-on azido; phenoxo oxygen and mu-CH3COO-1 kappa O center dot 2 kappa O' triple bridges to result in a tetranuclear unit Analyses of variable-temperature magnetic susceptibility data indicates that there is a global weak antiferromagnetic interaction between the copper(II) ions in complexes 1-3, with the exchange parameter J of -9 86, -11 6 and -19 98 cm(-1) for 1-3, respectively In complex 4 theoretical calculations show the presence of an antiferromagnetic coupling in the triple bridging ligands (acetato, phenoxo and azido) while the interaction through the double end-on azido bridging ligand is strongly ferromagnetic.

A hydrogen-1 nuclear magnetic resonance, mass spectral and extended Hückel study of some olefin complexes of rhodium(I) and iridium(I) and the crystal and molecular structure of η4-2,4-dimethylpenta-1,4-diene(η5-formylcyclopentadienyl)rhodium(I)

A 1H NMR study of monosubstituted η-cyclopentadienyl-rhodium(I) complexes of type LLRh(C5H4X) and -iridium(I) complexes of type L2Ir(C5H4X) (L = ethene, LL = 1,3- or 1,5-diolefin; X = C(C6H5)3, CHO, or COOCH3) has been carried out. For complexes of both metals in which the neutral ligand is ethene or a non-conjugated diolefin the NMR spectra of the cyclopentadienyl protons are unusual in that H(2), H(5) resonate to high field either at room temperature or below. The corresponding NMR spectra for the cyclopentadienyl ring protons of complexes where the neutral ligand is a conjugated diene are, with one exception, normal. A single crystal X-ray structural analysis of (η4-2,4-dimethylpenta-1,4-diene)(η5-formylcyclopentadienyl)rhodium(I) (which exhibits an abnormal 1H NMR spectrum) reveals substantial localisation of electron density in the C(3)C(4) Cp ring bond (1.283(33) Å) which may be consistent with a contribution from an ‘allyl-ene’ rotamer to the ring—metal bonding scheme. An extended Hückel calculation with self consistent charge iteration was performed on this complex. The results predict a greater Mulliken overlap population for the C(3)C(4) bond in the cyclopentadienyl ring and show that the localisation is dependent on both the Cp ring substituent and the nature of the diolefin. The mass spectral fragmentation patterns of some representative diene complexes of iridium(I) and rhodium(I) are presented.

Do the legs of magnetic clouds contain twisted flux-rope magnetic fields?

Magnetic clouds (MCs) are a subset of interplanetary coronal mass ejections (ICMEs) characterised primarily by a smooth rotation in the magnetic field direction indicative of the presence of a magnetic flux rope. Energetic particle signatures suggest MC flux ropes remain magnetically connected to the Sun at both ends, leading to widely used model of global MC structure as an extended flux rope, with a loop-like axis stretching out from the Sun into the heliosphere and back to the Sun. The time of flight of energetic particles, however, suggests shorter magnetic field line lengths than such a continuous twisted flux rope would produce. In this study, two simple models are compared with observed flux rope axis orientations of 196 MCs to show that the flux rope structure is confined to the MC leading edge. The magnetic cloud “legs,” which magnetically connect the flux rope to the Sun, are not recognisable as MCs and thus are unlikely to contain twisted flux rope fields. Spacecraft encounters with these non-flux rope legs may provide an explanation for the frequent observation of non-magnetic cloud ICMEs.

Magnetic Susceptibility Measurements at Ultra-low Temperatures

We report the design and operation of a device for ac magnetic susceptibility measurements that can operate down to 1 mK. The device, a modification of the standard mutual inductance bridge, is designed with detailed consideration of the thermalization and optimization of each element. First, in order to reduce local heating, the primary coil is made with superconducting wire. Second, a low-temperature transformer which is thermally anchored to the mixing chamber of a dilution refrigerator, is used to match the output of the secondary coil to a high-sensitivity bridge detector. The careful thermal anchoring of the secondary coil and the matching transformer is required to reduce the overall noise temperature and maximize sensitivity. The sample is immersed in liquid (3)He to minimize the Kapitza thermal resistance. The magnetic susceptibility of several magnetic compounds, such as the well-known spin gap compound NiCl(2)-4SC(NH(2))(2) and other powdered samples, have been successfully measured to temperatures well below 10 mK.

Nuclear Magnetic Resonance Study of Hydrated Bentonite

Impedance spectroscopy and nuclear magnetic resonance (NMR) were used to investigate the mobility of water molecules located in the interlayer space of H(+) - exchanged bentonite clay. The conductivity obtained by ac measurements was 1.25 x 10(-4) S/cm at 298 K. Proton ((1)H) lineshapes and spin-lattice relaxation times were measured as a function of temperature over the temperature range 130-320 K. The NMR experiments exhibit the qualitative features associated with the proton motion, namely the presence of a (1)H NMR line narrowing and a well-defined spin-lattice relaxation rate maximum. The temperature dependence of the proton spin-lattice relaxation rates was analyzed with the spectral density function appropriate for proton dynamics in a two-dimensional system. The self-diffusion coefficient estimated from our NMR data, D similar to 2 x 10(-7) cm(2)/s at 300 K, is consistent with those reported for exchanged montmorillonite clay hydrates studied by NMR and quasi-elastic neutron scattering (QNS).