Direct methods and residue type specific isotope labeling in NMR structure determination and model-driven sequential assignment

Direct methods in NMR based structure determination start from an unassigned ensemble of unconnected gaseous hydrogen atoms. Under favorable conditions they can produce low resolution structures of proteins. Usually a prohibitively large number of NOEs is required, to solve a protein structure ab-initio, but even with a much smaller set of distance restraints low resolution models can be obtained which resemble a protein fold. One problem is that at such low resolution and in the absence of a force field it is impossible to distinguish the correct protein fold from its mirror image. In a hybrid approach these ambiguous models have the potential to aid in the process of sequential backbone chemical shift assignment when ¹³C^β and ¹³C′ shifts are not available for sensitivity reasons. Regardless of the overall fold they enhance the information content of the NOE spectra. These, combined with residue specific labeling and minimal triple-resonance data using ¹³C^α connectivity can provide almost complete sequential assignment. Strategies for residue type specific labeling with customized isotope labeling patterns are of great advantage in this context. Furthermore, this approach is to some extent error-tolerant with respect to data incompleteness, limited precision of the peak picking, and structural errors caused by misassignment of NOEs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Tuning the HNN experiment: generation of serine–threonine check points

We describe here the tunability of the HNN experiment to obtain certain residue specific peak patterns in the spectra of (¹⁵N, ¹³C) labeled proteins. This is achieved by tuning a band-selective 180° pulse on the carbon channel in the pulse sequence, whereby one can tamper with the C^α–C^β coupling evolutions for the different residues. Specifically, we generate distinctive peak patterns for serine and threonine and their neighbors in the different planes of the three dimensional spectrum. These provide useful anchor points during sequential assignment of backbone resonances. The performance of this experiment, referred to as HNN-ST here, is demonstrated using two proteins, one properly folded and the other completely denatured. With the availability of high field spectrometers, techniques such as TROSY, and ever increasing sensitivities in the probes, this experiment with its large number of check points has a great potential for rapid and unambiguous backbone resonance assignment in large proteins.     

Band-selective 13C Homonuclear 3D Spectroscopy for Solid Proteins at High Field with Rotor-synchronized Soft Pulses

We demonstrate improved 3D ¹³C–¹³C–¹³C chemical shift correlation experiments for solid proteins, utilizing band-selective coherence transfer, scalar decoupling and homonuclear zero-quantum polarization transfer. Judicious use of selective pulses and a z-filter period suppress artifacts with a two-step phase cycle, allowing higher digital resolution in a fixed measurement time. The novel correlation of C^ali–C^ali–CX (C^ali for aliphatic carbons, CX for any carbon) reduces measurement time by an order of magnitude without sacrificing digital resolution. The experiment retains intensity from side-chain carbon resonances whose chemical shift dispersion is critical to minimize spectral degeneracy for large proteins with a predominance of secondary structure, such as β-sheet rich fibrillar proteins and α-helical membrane proteins. We demonstrate the experiment for the β1 immunoglobulin binding domain of protein G (GB1) and fibrils of the A30P mutant of α-synuclein, which is implicated in Parkinson’s disease. Selective pulses of duration comparable the rotor period give optimal performance, but must be synchronized with the spinning in non-trivial ways to minimize chemical shift anisotropy recoupling effects. Soft pulses with a small bandwidth-duration product are best for exciting the ~70 ppm bandwidth required for aliphatic-only dimensions.     

Dysregulation of neural calcium signaling in Alzheimer disease,bipolar disorder and schizophrenia

Neurons have highly developed Ca²⁺ signaling systems responsible for regulating a large number of neural functions such as the control of brain rhythms, information processing and the changes in synaptic plasticity that underpin learning and memory. The tonic excitatory drive, which is activated by the ascending arousal system, is particularly important for processes such as sensory perception, cognition and consciousness. The Ca²⁺ signaling pathway is a key component of this arousal system that regulates the neuronal excitability responsible for controlling the neural brain rhythms required for information processing and cognition. Dysregulation of the Ca²⁺ signaling pathway responsible for many of these neuronal processes has been implicated in the development of some of the major neural diseases in man such as Alzheimer disease, bipolar disorder and schizophrenia. Various treatments, which are known to act by reducing the activity of Ca²⁺ signaling, have proved successful in alleviating the symptoms of some of these neural diseases.     

Reliable short-term memory in the trion model: toward a cortical language and grammar

 It has previously been shown that Hebb learning in a single column in the trion model of cortical organization occurs by selection. Motivated by von Neumann's solution for obtaining reliability and by models of circulating cortical activity, we introduce Hebb intercolumnar couplings to achieve dramatic enhancements in reliability in the firing of connected columns. In order for these enhancements to occur, specific temporal phase differences must exist between the same inherent spatial-temporal memory patterns in connected columns. We then generalize the criteria of large enhancements in the global firing of the entire connected columnar network to investigate the case when different inherent memory patterns are in the columns. The spatial rotations as well as the temporal phases now are crucial. Only certain combinations of inherent memory patterns meet these criteria with the symmetry properties playing a major role. The columnar order of these memory patterns not in the same symmetry family can be extremely important. This yields the first higher-level architecture of a cortical language and grammar within the trion model. The implications of this result with regard to an innate human language and grammar are discussed. Received: 14 June 2000 / Accepted in revised form: 25 July 2000     

Influence of synthetic packing materials on the gas dispersion and biodegradation kinetics in fungal air biofilters

The biodegradation of toluene was studied in two lab-scale air biofilters operated in parallel, packed respectively with perlite granules (PEG) and polyurethane foam cubes (PUC) and inoculated with the same toluene-degrading fungus. Differences on the material pore size, from micrometres in PEG to millimetres in PUC, were responsible for distinct biomass growth patterns. A compact biofilm was formed around PEG, being the interstitial spaces progressively filled with biomass. Microbial growth concentrated at the core of PUC and the excess of biomass was washed-off, remaining the gas pressure drop comparatively low. Air dispersion in the bed was characterised by tracer studies and modelled as a series of completely stirred tanks (CSTR). The obtained number of CSTR (n) in the PEG packing increased from 33 to 86 along with the applied gas flow (equivalent to empty bed retention times from 48 to 12 s) and with operation time (up to 6 months). In the PUC bed, n varied between 9 and 13, indicating that a stronger and steadier gas dispersion was achieved. Michaelis–Menten half saturation constant (k _m) estimates ranged 71–113 mg m⁻³, depending on the experimental conditions, but such differences were not significant at a 95% confidence interval. The maximum volumetric elimination rate (r _m) varied from 23 to 50 g m⁻³ h⁻¹. Comparison between volumetric and biomass specific biodegradation activities indicated that toluene mass transfer was slower with PEG than with PUC as a consequence of a smaller biofilm surface and to the presence of larger zones of stagnant air.     

Combined effects of an oxidative enzyme and dissolved humic substances on 13C-labelled 2,4-D herbicide as revealed by high-resolution 13C NMR spectroscopy

Phenoxyalkanoic acids are a widely used class of herbicides. This work employed high-resolution ¹³C NMR to study the structural changes induced by humic substances and horseradish perodixase on 2,4-dichorophenoxyacetic acid (2,4-D) ¹³C-labelled in the side chain. NMR spectra showed that humic substances chemically catalyze abiotic splitting of [¹³C]2,4-D into 2,4-dichlorophenol and [¹³C]acetic acid at pH 7 but not at pH 4.7. Peroxidase did not catalyze the oxidative degradation of [¹³C]2,4-D at any pH tested and inhibited the effect of humic substances. Catalytic degradation by humic substances was attributed to free-radical reactions enhanced by the stereochemical contribution of large conformational structures formed by heterogeneous humic molecules at neutral pHs. Inhibition of 2,4-D degradation when humic substances were combined with peroxidase was explained by modification of both chemical and conformational humic structure due to peroxidase-promoted oxidative cross-coupling among humic molecules. Our findings show for the first time that the abiotic degradation of 2,4-D is catalyzed by dissolved humic substances at neutral pH. Journal of Industrial Microbiology & Biotechnology (2001) 26, 70–76. Received 09 February 2000/ Accepted in revised form 22 May 2000     

Calcium Signalling and Alzheimer’s Disease

New insights into how Ca²⁺ regulates learning and memory have begun to provide clues as to how the amyloid-dependent remodelling of neuronal Ca²⁺ signalling pathways can disrupt the mechanisms of learning and memory in Alzheimer’s disease (AD). The calcium hypothesis of AD proposes that activation of the amyloidogenic pathway remodels the neuronal Ca²⁺ signalling pathways responsible for cognition by enhancing the entry of Ca²⁺ and/or the release of internal Ca²⁺ by ryanodine receptors or InsP₃ receptors. The specific proposal is that Ca²⁺ signalling remodelling results in a persistent elevation in the level of Ca²⁺ that constantly erases newly acquired memories by enhancing the mechanism of long-term depression (LTD). Neurons can still form memories through the process of LTP, but this stored information is rapidly removed by the persistent activation of LTD. Further dysregulation in Ca²⁺ signalling will then go on to induce the neurodegeneration that characterizes the later stages of dementia.     

Reduction of phenolics content and COD in olive oil mill wastewaters by indigenous yeasts and fungi

Olive oil mill wastewaters (OOMW) cause a recurrent environmental pollution problem. The large concentration of phenolic compounds in the organic fraction of OOMW is principally responsible for the phytotoxicity and microbial growth inhibitory effects of the effluent. Candida boidinii, Geotrichum candidum, a Penicillium sp. and Aspergillus niger HA37 were isolated from OOMW. When cultivated directly on an undiluted OOMW-based medium containing 82 g l⁻¹ COD, these strains removed only 4–8% of chemical oxygen demand (COD) and phenolics. In contrast, reduction values attaining respectively 40–73% for phenolics and 45–78% for COD removal in the undiluted OOMW-based medium were obtained when using the strains gradually acclimated to high concentration of OOMW by successive stepwise transfer from media containing COD of 20.5 up to 82 g l⁻¹. Possibly, a sufficient production level of degradation and/or detoxification enzymes has to be attained to overcome the toxic effects of the phenolic fraction of concentrated OOMW. The present investigation calls attention to the necessity of acclimation for certain fungal and yeasts strains potentially useful for treating highly polluted effluents.     

Impact of target site distribution for Type I restriction enzymes on the evolution of methicillin-resistant Staphylococcus aureus (MRSA) populations

A limited number of Methicillin-resistant Staphylococcus aureus (MRSA) clones are responsible for MRSA infections worldwide, and those of different lineages carry unique Type I restriction-modification (RM) variants. We have identified the specific DNA sequence targets for the dominant MRSA lineages CC1, CC5, CC8 and ST239. We experimentally demonstrate that this RM system is sufficient to block horizontal gene transfer between clinically important MRSA, confirming the bioinformatic evidence that each lineage is evolving independently. Target sites are distributed randomly in S. aureus genomes, except in a set of large conjugative plasmids encoding resistance genes that show evidence of spreading between two successful MRSA lineages. This analysis of the identification and distribution of target sites explains evolutionary patterns in a pathogenic bacterium. We show that a lack of specific target sites enables plasmids to evade the Type I RM system thereby contributing to the evolution of increasingly resistant community and hospital MRSA.     

Flexible Memory Networks

Networks of neurons in some brain areas are flexible enough to encode new memories quickly. Using a standard firing rate model of recurrent networks, we develop a theory of flexible memory networks. Our main results characterize networks having the maximal number of flexible memory patterns, given a constraint graph on the network’s connectivity matrix. Modulo a mild topological condition, we find a close connection between maximally flexible networks and rank 1 matrices. The topological condition is H ₁(X;ℤ)=0, where X is the clique complex associated to the network’s constraint graph; this condition is generically satisfied for large random networks that are not overly sparse. In order to prove our main results, we develop some matrix-theoretic tools and present them in a self-contained section independent of the neuroscience context.     

Models for Proton-Coupled Electron Transfer in Photosystem II

The coupling of proton and electron transfers is a key part of the chemistry of photosynthesis. The oxidative side of photosystem II (PS II) in particular seems to involve a number of proton-coupled electron transfer (PCET) steps in the S-state transitions. This mini-review presents an overview of recent studies of PCET model systems in the authors’ laboratory. PCET is defined as a chemical reaction involving concerted transfer of one electron and one proton. These are thus distinguished from stepwise pathways involving initial electron transfer (ET) or initial proton transfer (PT). Hydrogen atom transfer (HAT) reactions are one class of PCET, in which H⁺ and e ⁻ are transferred from one reagent to another: AH+B→A+BH, roughly along the same path. Rate constants for many HAT reactions are found to be well predicted by the thermochemistry of hydrogen transfer and by Marcus Theory. This includes organic HAT reactions and reactions of iron-tris(α-diimine) and manganese-(μ-oxo) complexes. In PS II, HAT has been proposed as the mechanism by which the tyrosine Z radical (Y_Z) oxidizes the manganese cluster (the oxygen evolving complex, OEC). Another class of PCET reactions involves transfer of H⁺ and e ⁻ in different directions, for instance when the proton and electron acceptors are different reagents, as in AH–B+C⁺→A–HB⁺+C. The oxidation of Y_Z by the chlorophyll P680 + has been suggested to occur by this mechanism. Models for this process – the oxidation of phenols with a pendent base – are described. The oxidation of the OEC by Y_Z could also occur by this second class of PCET reactions, involving an Mn–O–H fragment of the OEC. Initial attempts to model such a process using ruthenium-aquo complexes are described. An erratum to this article can be found at     

Optimization of amino acid type-specific <Superscript>13</Superscript>C and <Superscript>15</Superscript>N labeling for the backbone assignment of membrane proteins by solution- and solid-state NMR with the UPLABEL algorithm

We present a computational method for finding optimal labeling patterns for the backbone assignment of membrane proteins and other large proteins that cannot be assigned by conventional strategies. Following the approach of Kainosho and Tsuji (Biochemistry 21:6273–6279 (1982)), types of amino acids are labeled with ¹³C or/and ¹⁵N such that cross peaks between ¹³CO(i – 1) and ¹⁵NH(i) result only for pairs of sequentially adjacent amino acids of which the first is labeled with ¹³C and the second with ¹⁵N. In this way, unambiguous sequence-specific assignments can be obtained for unique pairs of amino acids that occur exactly once in the sequence of the protein. To be practical, it is crucial to limit the number of differently labeled protein samples that have to be prepared while obtaining an optimal extent of labeled unique amino acid pairs. Our computer algorithm UPLABEL for optimal unique pair labeling, implemented in the program CYANA and in a standalone program, and also available through a web portal, uses combinatorial optimization to find for a given amino acid sequence labeling patterns that maximize the number of unique pair assignments with a minimal number of differently labeled protein samples. Various auxiliary conditions, including labeled amino acid availability and price, previously known partial assignments, and sequence regions of particular interest can be taken into account when determining optimal amino acid type-specific labeling patterns. The method is illustrated for the assignment of the human G-protein coupled receptor bradykinin B2 (B₂R) and applied as a starting point for the backbone assignment of the membrane protein proteorhodopsin.     

Interpretation of nitrogen isotope signatures using the NIFTE model

Nitrogen cycling in forest soils has been intensively studied for many years because nitrogen is often the limiting nutrient for forest growth. Complex interactions between soil, microbes, and plants and the consequent inability to correlate δ¹⁵N changes with biologic processes have limited the use of natural abundances of nitrogen isotopes to study nitrogen (N) dynamics. During an investigation of N dynamics along the 250-year-old successional sequence in Glacier Bay, Alaska, United States, we observed several puzzling isotopic patterns, including a consistent decline in δ¹⁵N of the late successional dominant Picea at older sites, a lack of agreement between mineral N δ¹⁵N and foliar δ¹⁵N, and high isotopic signatures for mycorrhizal fungi. In order to understand the mechanisms creating these patterns, we developed a model of N dynamics and N isotopes (Nitrogen Isotope Fluxes in Terrestrial Ecosystems, NIFTE), which simulated the major transformations of the N cycle and predicted isotopic signatures of different plant species and soil pools. Comparisons with field data from five sites along the successional sequence indicated that NIFTE can duplicate observed patterns in δ¹⁵N of soil, foliage, and mineral N over time. Different scenarios that could account for the observed isotopic patterns were tested in model simulations. Possible mechanisms included increased isotopic fractionation on mineralization, fractionation during the transfer of nitrogen from mycorrhizal fungi to plants, variable fractionation on uptake by mycorrhizal fungi compared to plants, no fractionation on mycorrhizal transfer, and elimination of mycorrhizal fungi as a pool in the model. The model results suggest that fractionation during mineralization must be small (˜2‰), and that no fractionation occurs during plant or mycorrhizal uptake. A net fractionation during mycorrhizal transfer of nitrogen to vegetation provided the best fit to isotopic data on mineral N, plants, soils, and mycorrhizal fungi. The model and field results indicate that the importance of mycorrhizal fungi to N uptake is probably less under conditions of high N availability. Use of this model should encourage a more rigorous assessment of isotopic signatures in ecosystem studies and provide insights into the biologic transformations which affect those signatures. This should lead to an enhanced understanding of some of the fundamental controls on nitrogen dynamics. Received: 1 July 1998 / Accepted: 23 December 1998     

Exhaust gas purification using immobilised monocultures (biocatalysts)

Summary The paper is concerned with the purification of exhaust gases using biocatalysts in a trickle bed reactor. Substance specific strains (monocultures) which were, for example, immobilised on activated carbon served as biocatalyst. Technically important solvents and substances such as aldehydes, methyl ethyl ketone and ethyl acetate were used as pollutants. Their concentration was about 5–40 ppm in the exhaust gas to be purified. The experiments show that with suitable bacterial, strains space velocities of about k ^*=1500 h^-1 can be obtained at a conversion of 90%. The mass transfer through the liquid film around the activated carbon grains seems to be rate determining.     

Interpretation of fluorescence decay kinetics in 3-methylbenzimidazolyl(5′-5′)guanosine dinucleotides: exponential dependence on the number of phosphates in the polyphosphate bridge

The number of phosphate groups in the 5′,5′-polyphosphate bridge of mRNA-cap dinucleotide analogues affects kinetics of long-range electron transfer (ET) responsible for 3-methylbenzimidazole (m³B) fluorescence quenching in model dinucleotides. For instance, 3-methylbenzimidazolyl(5′-5′)guanosine dinucleotides (m³Bp _n G, n = 2, 3, 4) having m³B donor, 5′-5′ polyphosphate bridge, and guanine (G) acceptor, exhibit exponential dependence of the ET rate on the number of phosphates, i.e. donor–acceptor distance. Involvement of the 5′-5′ polyphosphate bridge in the ET is strongly indicated by lack of m³B-G stacking effect on the exponential factor, which is the same at 20°C, where m³B-G intramolecular stacking dominates, as that at 75°C where stacking–unstacking equilibrium is shifted in favour of the unstacked structure.     

Effects of superoxide radicals on ACC synthase activity in chilling-stressed etiolated mungbean seedlings

The activity of 1-aminocyclopropane-1-carboxylic acid synthase (ACC synthase, ACS) and the concentrations of superoxide radical (O₂^−.) and hydrogen peroxide (H₂O₂) were measured in etiolated mungbean seedlings following their transfer to a growth chamber at 25°C after a 5-h-chilling treatment at 5°C. All of these variables increased dramatically after the transfer, and strong correlations were found between ACS activity and the concentrations of superoxide and H₂O₂. Exogenous applications of two generators of superoxide radicals, methylviologen (MV) and xanthine–xanthine oxidase (X–XOD), enhanced ACS activity in seedlings, but their effects were inhibited by exogenous applications of specific scavengers of O₂^−.. However, applications of H₂O₂ or specific H₂O₂-scavengers had no significant effects on seedlings ACS activity. The results indicate that O₂^−. was involved in the chilling-induced increases in ACS activity, but not H₂O₂. ACS activity peaked ca. 8 h after the transfer, and then declined, but the decline could be counteracted by exogenous applications of specific O₂^−. scavengers, this suggests that damage was caused by superoxide radicals influencing ACS activity in etiolated mungbean seedlings. Further analysis of changes in two key kinetic parameters of ACS activity—V _max (maximum velocity) and K _m (the Michaelis constant)—in the seedlings indicated that the presence of O₂^−. may reduce K _m, i.e. increase substrate (S-adenosyl methionine, SAM) affinity. That would be the main mechanism responsible for the observed chilling-induced increases in ACS activity in etiolated mungbean seedlings.     

The effects of arginine vasotocin and catecholamines on nitrogen excretion and the cardio-respiratory physiology of the gulf toadfish, Opsanus beta

Simultaneous measurements of cardio-respiratory variables, oxygen uptake and whole body urea/ammonia/tritiated water effluxes were performed on cannulated gulf toadfish, Opsanus beta, before and after intra-arterial injection of the vasoactive agents, adrenaline, isoproterenol and arginine vasotocin. These experiments were conducted to test the hypothesis that the phenomenon of pulsatile urea excretion might reflect sudden changes in the general diffusive properties of the gill for solute transfer. Injection of isoproterenol (final nominal circulating level = 10⁻⁶ mol l⁻¹), was used as a tool to maximise the diffusive and perfusive conditions for branchial solute transfer. This protocol caused a pronounced reduction in arterial blood pressure, an elevation of cardiac frequency and associated increases in whole body urea and tritiated water effluxes; ammonia excretion and oxygen uptake were unaffected. Injection of adrenaline (final nominal circulating level=10⁻⁶ mol l⁻¹), caused a significant increase in arterial blood pressure and a tachycardia, yet nitrogen excretion and oxygen uptake were unaffected. Injection of arginine vasotocin, caused a dose-dependent (final nominal circulating levels = 10⁻¹¹–10⁻⁹ mol l⁻¹) increase in arterial blood pressure without affecting cardiac or ventilation frequency. At the two higher concentrations, arginine vasotocin caused large and transient increases in urea excretion without significantly affecting ammonia, water or oxygen fluxes. These results suggest that increased gill diffusive or perfusive conductance, while capable of augmenting urea efflux, cannot fully explain the sudden and massive increases in urea transfer associated with pulsatile urea excretion in toadfish. It is suggested that pulsatile urea excretion in this species may reflect a specific enhancement of urea excretion under the control of the neurohypophyseal hormone, arginine vasotocin. Accepted: 21 April 1998     

Stable isotopic structure of aquatic ecosystems

Isotopic, biogeochemical and ecological structure can provide a new dimension for understanding material flows, and the simultaneous function and structure of an ecosystem. Distributions ofδ ¹³C andδ ¹⁵N for biogenic substances in the Nanakita river estuary involving Gamo lagoon in Japan were investigated to construct isotope biogeochemical and ecological structure for assessing fate and transfer of organic matter, and food web structure. The isotopic framework of the ecosystem was successfully described in aδ ¹⁵N–δ ¹³C map. In this estuary the variations of isotope ratios of biogenic substances were clearly explained by the mixing of land-derived organic matter, and marine-derived organic matter. A trophic-level effect of¹⁵N enrichment was clearly observed. Organisms were classified into three groups depending upon the contribution of land-derived organic matter in a food chain. Almost all biota except mollusca in the lagoon depend on organic matter of marine origin. The contributions of both land and marine organic matter were comparable for mollusca in the lagoon.