Structure and tunneling splitting spectra of methyl groups of tetramethylpyrazine in complexes with chloranilic and bromanilic acids.

The crystal and molecular structure of the 2,3,5,6-tetramethylpyrazine (TMP) complex with 2,5-dibromo-3,6-dihydroxy-p-quinone (bromanilic acid, BRA) has been studied and the results are compared with TMP CLA (2,5-dichloro-3,6-dihydroxy-p-quinone (chloranilic acid, CLA) complex. The X-ray structure of TMP BRA complex indicates the formation of dimeric units, in which two BRA(-) anions are connected by two O-H···O (2.646(2) Å) hydrogen bonds, whereas the cations and anions are joined together by strong N(+)-H···O(-) (2.657(2) Å) hydrogen bonds. The results are analyzed in terms of both the methyl group surroundings and the C-H···O and N(+)-H···O(-) (or N···H-O) bridge formations. Both effects, the strength of the N(+)-H···O(-) hydrogen bonds and steric hindrance for the rotations, are responsible for the CH3 group dynamics. For the TMP CLA and TMP BRA complexes, the inelastic neutron backscattering spectra were also investigated. In the case of TMP CLA, four tunneling signals have been observed in the energy range ±30 µeV, which indicates four inequivalent methyl groups in the crystal structure at the lowest temperature. No tunneling splitting is observed in the case of the TMP BRA complex, most probably due to the overlapping with the elastic peak. The tunneling results are consistent with the (1)H NMR spin-lattice relaxation time investigations in a wide temperature range, which also point to the CH3 group tunneling effect in the case of TMP CLA.

On the cooperative nature of the ß-process in neat and binary glasses: a dielectric and nuclear magnetic resonance spectroscopy study.

By means of dielectric as well as (2)H and (31)P nuclear magnetic resonance spectroscopy (NMR) the component dynamics of the binary glass tripropyl phosphate (TPP)/polystyrene (PS/PS-d3) is selectively investigated for concentrations distributed over the full range. We study the secondary (ß-) relaxation below T(g), which is found in all investigated samples containing TPP, but not in neat polystyrene. The dielectric spectrum of the ß-process is described by an asymmetric distribution of activation energies, essentially not changing in the entire concentration regime; its most probable value is E/k ≅ 24 T(g). Persistence of the ß-process is confirmed by (31)P NMR Hahn-echo and spin-lattice relaxation experiments on TPP, which identify the nature of the ß-process as being highly spatially hindered as found for other (neat) glasses studied previously, or re-investigated within this work. The corresponding (2)H NMR experiments on PS-d3 confirm the absence of a ß-process in neat PS-d3, but reveal a clear signature of a ß-process in the mixture, i.e., polystyrene monomers perform essentially the same type of secondary relaxation as the TPP molecules. Yet, there are indications that some fractions of PS-d3 as well as TPP molecules become immobilized in the mixture in contrast to the case of neat glasses. We conclude that in a binary glass the ß-process introduced by one component induces a highly similar motion in the second component, and this may be taken as an indication of its cooperative nature.

Microscopic dynamics of polyethylene glycol chains interacting with silica nanoparticles.

We present high resolution neutron spectroscopic investigations of polyethylene glycol matrices interacting attractively with neat SiO2 nanoparticles. We observe a very rich dynamical picture that significantly contradicts earlier conclusions on such systems. Investigating a short chain matrix we realized that a fraction of chains is attached at the nanoparticle surface suppressing completely its translational diffusion. Nevertheless these attached chains undergo an unchanged segmental dynamics seemingly forming a micellelike corona of chains attached with their OH end groups. Changing to methyl-terminated chains the picture changes drastically, now showing a tightly adsorbed layer that however is not glassy as often assumed but undergoes fast picosecond local dynamics. With the singular importance of end groups, mean field approaches are not applicable and future simulations should be redirected to model such unexpected phenomena.

Thermal fluctuations of haemoglobin from different species: adaptation to temperature via conformational dynamics.

Thermodynamic stability, configurational motions and internal forces of haemoglobin (Hb) of three endotherms (platypus, Ornithorhynchus anatinus; domestic chicken, Gallus gallus domesticus and human, Homo sapiens) and an ectotherm (salt water crocodile, Crocodylus porosus) were investigated using circular dichroism, incoherent elastic neutron scattering and coarse-grained Brownian dynamics simulations. The experimental results from Hb solutions revealed a direct correlation between protein resilience, melting temperature and average body temperature of the different species on the 0.1 ns time scale. Molecular forces appeared to be adapted to permit conformational fluctuations with a root mean square displacement close to 1.2 Å at the corresponding average body temperature of the endotherms. Strong forces within crocodile Hb maintain the amplitudes of motion within a narrow limit over the entire temperature range in which the animal lives. In fully hydrated powder samples of human and chicken, Hb mean square displacements and effective force constants on the 1 ns time scale showed no differences over the whole temperature range from 10 to 300 K, in contrast to the solution case. A complementary result of the study, therefore, is that one hydration layer is not sufficient to activate all conformational fluctuations of Hb in the pico- to nanosecond time scale which might be relevant for biological function. Coarse-grained Brownian dynamics simulations permitted to explore residue-specific effects. They indicated that temperature sensing of human and chicken Hb occurs mainly at residues lining internal cavities in the ß-subunits.

Strain induced anisotropies in silica polydimethylsiloxane composites.

Structural changes of silica in polydimethylsiloxane rubber induced by external forces were studied by means of small-angle X-ray scattering experiments. The silica fraction varies from 9 up to 23 vol% and the elongation ratio from 1 to 3. Within the q-range of 0.02 nm(-1)

Rotational dynamics and coupling of methyl group rotations in methyl fluoride studied by high resolution inelastic neutron scattering.

Methyl group rotations in methyl fluoride were studied using the high flux backscattering spectrometer SPHERES at FRM-II. The asymmetry and width of the low temperature tunneling peak was used to determine if coupled rotations between neighboring methyl fluoride molecules exist. The temperature dependent broadening of the tunneling peak was used to determine the first librational transition and compared to the temperature dependent shift of the position of the tunneling peak. The results obtained by using inelastic neutron scattering confirm previous models that assume rotational coupling. This is the first neutron backscattering experiment with sub-microeV resolution at energy transfers up to 31 microeV.

Direct evidence for the Nd magnetic ordering in NdMnO(3) from the hyperfine field splitting of Nd nuclear levels.

We investigated the low energy excitations in NdMnO(3) in the µeV range by a backscattering neutron spectrometer. The energy spectra on polycrystalline NdMnO(3) samples revealed inelastic peaks at E = 2.15 ± 0.01 µeV at T = 2 K on both energy gain and energy loss sides. The inelastic peaks move gradually towards lower energy with increasing temperature and tend to merge with the elastic peak at the electronic magnetic ordering temperature of Nd, T(Nd)≈20 K. However, at temperatures higher than T(Nd)≈20 K the energy of the inelastic peak decreases at a much slower rate and remains finite up to T = 55 K, the highest temperature investigated. We interpret the inelastic peaks to be due to the transition between the hyperfine-split nuclear level of the (143)Nd and (145)Nd isotopes with spin I = 7/2 caused by the magnetic ordering of Nd electronic moment below T(Nd)≈20 K. We ascribe the finite energy of the inelastic peak and its much smaller temperature dependence at T>20 K to be due to the polarization of the Nd magnetic moment by the field of Mn moments that order below T(N)≈78 K.

Anomalous hyperfine interaction in CoF(2) investigated by high resolution neutron spectroscopy.

We investigated the low energy excitations in CoF(2) in the µeV range with a back-scattering neutron spectrometer. The energy scans on a CoF(2) powder sample revealed inelastic peaks at E = 0.728 ± 0.008 µeV at T = 3.46 K on both energy gain and energy loss sides. The inelastic peaks move gradually towards lower energy with increasing temperature and finally merge with the elastic peak at the electronic magnetic ordering temperature T(N)≈37 K. We interpret the inelastic peaks to be due to the transition between the hyperfine-split nuclear level of the (59)Co isotopes with spin I = 7/2. We have shown that the energy of the inelastic peak or the hyperfine splitting in CoF(2) can be treated as an order parameter of the antiferromagnetic phase transition and yields the critical exponent ß = 0.313 ± 0.007, consistent with the neutron diffraction results and also the three-dimensional Ising character of the magnetic system. The determined hyperfine splitting in CoF(2) deviates from the linear relationship between the ordered electronic magnetic moment and the hyperfine splitting in Co, Co-P amorphous alloys and CoO, presumably due to the presence of an unquenched orbital moment.

An analysis of subunit exchange in the dimeric DNA-binding and DNA-bending protein, TF1.

TF1 is the Bacillus subtilis bacteriophage-encoded dimeric type II DNA-binding protein. This relative of the eubacterial HU proteins and of the Escherichia coli integration host factor binds preferentially to 5-(hydroxymethyluracil)-containing DNA. We have examined the dynamics of exchange of monomer subunits between molecules of dimeric TF1. The analysis takes advantage of the fact that replacement of phenylalanine with arginine at amino acid 61 in the beta-loop 'arm' of TF1 alters DNA-bending and -binding properties, generating DNA complexes with distinctively different mobilities in gel electrophoresis. New species of DNA-protein complexes were formed by mixtures of wild type and mutant TF1, reflecting the formation of heterodimeric TF1, and making the dynamics of monomer exchange between TF1 dimers accessible to a simple gel retardation analysis. Exchange was rapid at high protein concentrations, even at 0 degrees C, and is proposed to be capable of proceeding through an interaction of molecules of TF1 dimer rather than exclusively through dissociation into monomer subunits. Evidence suggesting that DNA-bound TF1 dimers do not exchange subunits readily is also presented.

DNA-bending properties of TF1.

Transcription factor 1 (TF1) is the Bacillus subtilis phage SPO1-encoded member of the family of DNA-binding proteins that includes Escherichia coli HU and integration host factor, IHF. A gel electrophoretic retardation method has been used to show that a TF1 dimer binding to one of its preferred sites in (5-hydroxymethyl)uracil (hmUra)-containing DNA sharply bends the latter. In fact, the DNA-bending properties of TF1 and E. coli IHF are indistinguishable. Substitutions at amino acid 61 in the DNA-binding "arm" of TF1 are known to affect DNA-binding affinity and site selectivity. Experiments described here show that these substitutions also affect DNA bending. The selectivity of TF1 binding is very greatly diminished and the affinity is reduced when hmUra is replaced in DNA by thymine (T). An extension of the gel retardation method that permits an analysis of DNA bending by non-specifically bound TF1 is proposed. Under the assumptions of this analysis, the reduced affinity of TF1 for T-containing DNA is shown to be associated with bending that is still sharp. The analysis of the TF1-DNA interaction has also been extended by hydroxyl radical (.OH) and methylation interference footprinting at two DNA sites. At each of these sites, and on each strand, TF1 strongly protects three segments of DNA from attack by OH. Patches of protected DNA are centered approximately ten base-pairs apart and fall on one side of the B-helix. Methylation in either the major or minor groove in the central ten base-pairs of the two TF1 binding sites quantitatively diminishes, but does not abolish, TF1 binding. We propose that multiple protein contacts allow DNA to wrap around the relatively small TF1 dimer, considerably deforming the DNA B-helix in the process.

Promoter recognition by the RNA polymerase from vegetative cells of the cyanobacterium Anabaena 7120.

The transcription start points (tsp) of seven genes of Anabaena 7120 were previously identified by S1 nuclease protection and primer extension experiments using RNA extracted from cells. In the present work, these tsp were confirmed, with one exception, by in vitro transcription using purified RNA polymerases of Anabaena 7120 and Escherichia coli, and crude extracts of Anabaena 7120 active in transcription. In all cases, the template for transcription consisted of closed circular plasmid DNA in which the putative promoter-containing fragment was cloned in front of a strong terminator, which resulted in defined 'pseudo-runoff' transcripts whose sizes correspond (with one exception) to those expected on the basis of the tsp determined for in vivo RNA. These results, together with others obtained with templates containing bacteriophage T4 or cyanophage N1 promoters, led to the conclusion that the principal Anabaena 7120 RNA polymerase prefers promoters whose sequence and spacing approximate that of the E. coli consensus promoter, and that the Anabaena 7120 genes expressed in vegetative cells, characterized to date, have relatively weak promoters.

TF1, a bacteriophage-specific DNA-binding and DNA-bending protein.

We review and discuss the biological and DNA-binding properties of the bacteriophage SPO1 transcription factor 1 (TF1), a DNA-binding protein belonging to the ubiquitous prokaryotic family of Type II DNA-binding proteins. We review recent information on the effects of certain mutations in TF1 on DNA-binding and on its ability to sharply bend DNA. We also compare the DNA-binding properties of the three best-studied type II DNA-binding proteins, Escherichia coli HU, E. coli integration host factor, and TF1, and discuss them in the context of the structure and properties of chromatin in prokaryotes.

Stoichiometry of DNA binding by the bacteriophage SP01-encoded type II DNA-binding protein TF1.

The stoichiometry of DNA binding by the bacteriophage SP01-encoded type II DNA-binding protein TF1 has been determined. 3H-Labeled TF1 was allowed to bind to a 32P-labeled DNA fragment containing a TF1 binding site. Multiple TF1-DNA complexes were resolved from each other and from unbound DNA by native gel electrophoresis. DNA-protein complexes were cut from polyacrylamide gels, and the amounts of 3H and 32P contained in each slice were measured. A ratio of 1.12 +/- 0.06 TF1 dimer/DNA molecule was calculated for the fastest-migrating TF1-DNA complex. We conclude that TF1 has a DNA-binding unit of one dimer. More slowly migrating complexes are apparently formed by serial addition of single TF1 dimers.

Deletion of a 55-kilobase-pair DNA element from the chromosome during heterocyst differentiation of Anabaena sp. strain PCC 7120.

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 produces terminally differentiated heterocysts in response to a lack of combined nitrogen. Heterocysts are found approximately every 10th cell along the filament and are morphologically and biochemically specialized for nitrogen fixation. At least two DNA rearrangements occur during heterocyst differentiation in Anabaena sp. strain PCC 7120, both the result of developmentally regulated site-specific recombination. The first is an 11-kilobase-pair (kb) deletion from within the 3' end of the nifD gene. The second rearrangement occurs near the nifS gene but has not been completely characterized. The DNA sequences found at the recombination sites for each of the two rearrangements show no similarity to each other. To determine the topology of the rearrangement near the nifS gene, cosmid libraries of vegetative-cell genomic DNA were constructed and used to clone the region of the chromosome involved in the rearrangement. Cosmid clones which spanned the DNA separating the two recombination sites that define the ends of the element were obtained. The restriction map of this region of the chromosome showed that the rearrangement was the deletion of a 55-kb DNA element from the heterocyst chromosome. The excised DNA was neither degraded nor amplified, and its function, if any, is unknown. The 55-kb element was not detectably transcribed in either vegetative cells or heterocysts. The deletion resulted in placement of the rbcLS operon about 10 kb from the nifS gene on the chromosome. Although the nifD 11-kb and nifS 55-kb rearrangements both occurred under normal aerobic heterocyst-inducing conditions, only the 55-kb excision occurred in argon-bubbled cultures, indicating that the two DNA rearrangements can be regulated differently.

Characterization of two early promoters of cyanophage N-1.

Restriction fragments of cyanophage N-1 DNA, containing genes transcribed early in infection of Anabaena, were identified by hybridization with RNA prepared from infected cells. Two of these fragments were isolated by binding to Anabaena RNA polymerase on filters and were subsequently cloned. The start sites for transcription by Anabaena RNA polymerase were determined by run-off assays. The sequences of the two promoter regions thus localized were similar to each other in the -35 and -10 regions and nearly identical to the consensus sequence for promoters in Escherichia coli.

RNA polymerase subunit homology among cyanobacteria, other eubacteria and archaebacteria.

RNA polymerase purified from vegetative cells of the cyanobacterium Anabaena sp. strain PCC 7120 contains a dissociable sigma factor and a core of five subunits: the beta', beta, and two alpha subunits characteristic of all eubacteria and an additional 66,000-molecular-weight polypeptide called gamma. Fifteen of fifteen strains of unicellular and filamentous cyanobacteria tested contained a serologically related gamma protein. Antiserum to gamma reacted with Escherichia coli beta' and the A subunit of RNA polymerase of the archaebacterium Sulfolobus acidocaldarius. Thus the evolution of the RNA polymerase beta' subunit has followed different paths in three groups of procaryotes: cyanobacteria, other eubacteria, and archaebacteria.

Purification and characterization of RNA polymerase from the cyanobacterium Anabaena 7120.

A procedure for the purification of RNA polymerase from vegetative cells of the filamentous cyanobacterium Anabaena 7120 is described. Polyethyleneimine precipitation followed by gel filtration and affinity chromatography steps results in greater than 99% purification with 46% yield. The enzyme has a novel core component of Mr = 66,000, designated gamma, in addition to the typical prokaryotic beta'beta alpha 2 core enzyme. The sigma subunit has been identified by reconstitution of specific transcriptional activity from core enzyme and gel-purified sigma. In transcription assays, this RNA polymerase initiates at a number of Anabaena vegetative cell promoters, as well as from a bacteriophage T4 early promoter, but does not initiate at nitrogen fixation (nif) promoters used in heterocysts. The promoter specificity of Anabaena RNA polymerase is compared with that of Escherichia coli RNA polymerase.

Numerical, morphological and topographical heterogeneity of the chondriome during the vegetative life cycle of Polytoma papillatum.

Numerical, morphological, and topographical changes in the mitochondrial inventory (= chondriome) during the vegetative (= asexual) life cycle of Polytoma papillatum were examined by means of the serial-sectioning technique. At the onset of interphase the chondriome consists of primarily of one highly reticulated basket-shaped complex, which lines the periphery of the cell. Up to 3 additional fragments could be analysed, which, however, were much smaller and either spherical to ovoid, or elongated and poorly branched. During interphase growth both the size of the mitochondrial basket and the number of additional fragments (ca. 40) increases. During mitosis I (I. division cycle) the multiplication of the number of mitochondria gradually continues; the maximum (ca. 250) was counted at late telephase I and early cytokinesis I, but it cannot be excluded that the sparser short-lived meta- and anaphase cells, which were unfortunately missed, contain an even higher number of mitochondrial units. Concomitant with mitosis I, the mitochondrial basket is subdivided into several fragments; at the same time the mitochondria tend to form clusters and to occupy no longer merely peripheral but also more central regions. An irregular distribution of the chondriome results, with either local accumulations or local absence of mitochondria within definite regions of the cell. After completion of cytokinesis I the total number of mitochondria (ca. 80) is drastically reduced but it seems to increase again during the 2. division cycle. Irrespective of the number of preceding division cycles, the chondriome of young daughter cells enclosed in the sporangium usually resembles that of young interphase cells. Our results are discussed in relation to some of the current problems on mitochondriogenesis.

Morphogenesis of the plastidome and the flagellar apparatus during the vegetative life cycle of the colourless phytoflagellate Polytoma papillatum.

Serial-section reconstructions were used to study changes in morphology of the leucoplast and of flagellar apparatus during the vegetative life cycle of Polytoma papillatum. During interphase growth the volume of highly perforated leucoplast nearly doubled. The leucoplast of late interphase cells still had a deep cup shape (cyathiform), but several structural modifications were observed compared to the leucoplast of young interphase cells: either a thin and less perforated sheet-like area, taking up ca 25% of the external surface, and several double-layered areas occur sporadically at the tube-shaped part of the cup, or a bladder-shaped structure consisting of several very thin concentrically ringed layers occurred at the base of the tube. During preprophase the cyathiform leucoplast was transformed into a massive bowl-shaped (= poculiform) leucoplast, which was also characterized by thin-layered bladder-like or concentrically-ringed structures and by duplications in the form of pockets or chambers. The bipartition of the poculiform leucoplast occurred by furrowing, which started at preprophase and was completed only at the end of cytokinesis. The dynamics of the processes involved in these configural changes are discussed. During preprophase of the first division cycle the flagellar apparatus was subjected to several changes. The apical papilla which gave rise to the flagella was tied off into small 'extracytoplasmic' vesicles. This event caused a change in the orientation of the flagella to one another; the angle of ca 140 degrees during interphase was reduced to ca 90 degrees. The basal bodies detached from their axonemes and both the striated connection and the roots disintegrated. During prophase of the second division cycle the separation of the four basal bodies into two pairs and their migration towards the poles of the nucleus was completed; the outgrowth of the new daughter flagella also started. During late cytokinesis of the second division cycle the basal bodies had already duplicated and resynthesis of the striated connection was completed, whereas resynthesis of the roots was still in progress. The results are compared with those obtained from investigation of other algae.

The changes in ultrastructure during fertilization of the colourless flagellate Polytoma papillatum with special reference to the configural changes of their mitochondria.

Changes in the morphology of the mitochondrial inventory (= chondriome), the nucleus and the flagellar apparatus during the generative (sexual) life cycle of Polytoma papillatum were examined by means of the serial sectioning technique. At the onset of copulation gametes do not differ obviously from interphase cells of the vegetative (asexual) life cycle, in that, both primarily contain one basket-shaped mitochondrion. The quadriflagellate and binucleate zygote exhibits a chondriome which consists of one large highly reticulated basket at the periphery of the zygote and 33 smaller mitochondrial units. Therefore, the basket clearly results from fusion of the two gamete chondriomes. The smaller mitochondrial fragments are either spherical to ovoid or elongated and poorly branched; they tend to occupy more central regions of the zygote. After karyogamy the mitochondrial basket disintegrates into several fragments of various shapes and sizes. Most of the mitochondrial fragments are located at the periphery. At the onset of karyogamy the nuclei and the flagellar apparatuses do not differ significantly from those of the gametes and vegetative interphase cells. The diploid nucleus, however, is characterized by: 1. many spherical bodies (diameter: ca. 200 to 600 nm) which are found both in the nucleoplasm and in the nucleolus. The major part of these bodies consists of material whose ultrastructure resembles that of the "pars fibrosa" in the nucleolus; 2. three deep invaginations of the nuclear membrane, two of which extend to the nucleolus; 3. an increase of nucleoplasm-filled cavities in the nucleolar "pars granulosa". The four flagella are considerably shortened; the basal bodies bound to the flagella have lost their striated connection and the roots have nearly completely disappeared. The results are compared with those obtained from investigations in Chlamydomonas; their significance in extranuclear genetics and in the systematics of Volvocales is discussed.