A Stochastic Model of Cortical Microtubule Anchoring and Mechanics Provides Regulatory Control of Microtubule Shape.

The organization of cortical microtubule arrays play an important role in the development of plant cells. Until recently, the direct mechanical influence of cell geometry on the constrained microtubule (MT) trajectories have been largely ignored in computational models. Modelling MTs as thin elastic rods constrained on a surface, a previous study examined the deflection of MTs using a fixed number of segments and uniform segment lengths between MT anchors.

Baseline levels and characterization of hydrocarbons in surface marine sediments along the transportation corridor in Hudson Bay: A multivariate analysis of n-alkanes, PAHs and biomarkers.

Hudson Bay is a small arctic inland shelf sea which receives large amounts of freshwater from riverine discharges, with marine flow from the north and the Atlantic. A warming climate has resulted in an expanded open water season which will result in an increase in shipping of fuel oil and petroleum to communities and mines on the western shore, increasing the risk of hydrocarbon releases. To evaluate the status of hydrocarbons, surface sediments were collected at 34 locations in the transportation route and offshore and analysed for several types of hydrocarbons.

Saturation transfer difference NMR on the integral trimeric membrane transport protein GltPh determines cooperative substrate binding.

Saturation-transfer difference (STD) NMR spectroscopy is a fast and versatile method which can be applied for drug-screening purposes, allowing the determination of essential ligand binding affinities (K). Although widely employed to study soluble proteins, its use remains negligible for membrane proteins. Here the use of STD NMR for K determination is demonstrated for two competing substrates with very different binding affinities (low nanomolar to millimolar) for an integral membrane transport protein in both detergent-solubilised micelles and reconstituted proteoliposomes.

Helix N-Cap Residues Drive the Acid Unfolding That Is Essential in the Action of the Toxin Colicin A.

Numerous bacterial toxins and other virulence factors use low pH as a trigger to convert from water-soluble to membrane-inserted states. In the case of colicins, the pore-forming domain of colicin A (ColA-P) has been shown both to undergo a clear acidic unfolding transition and to require acidic lipids in the cytoplasmic membrane, whereas its close homologue colicin N shows neither behavior. Compared to that of ColN-P, the ColA-P primary structure reveals the replacement of several uncharged residues with aspartyl residues, which upon replacement with alanine induce an unfolded state at neutral pH.

Redox-dependent control of i-Motif DNA structure using copper cations.

Previous computational studies have shown that Cu+ can act as a substitute for H+ to support formation of cytosine (C) dimers with similar conformation to the hemi-protonated base pair found in i-motif DNA. Through a range of biophysical methods, we provide experimental evidence to support the hypothesis that Cu+ can mediate C-C base pairing in i-motif DNA and preserve i-motif structure. These effects can be reversed using a metal chelator, or exposure to ambient oxygen in the air that drives oxidation of Cu+ to Cu2+, a comparatively weak ligand.

Fixing the Conformation of Calix[4]arenes: When Are Three Carbons Not Enough?

Calix[4]arenes are unique macrocycles that through judicious functionalisation at the lower rim can be either fixed in one of four conformations or remain conformationally flexible. Introduction of propynyl or propenyl groups unexpectedly provides a new possibility; a unidirectional conformational switch, with the 1,3-alternate and 1,2-alternate conformers switching to the partial cone conformation, whilst the cone conformation is unchanged, under standard experimental conditions. Using H NMR kinetic studies, rates of switching have been shown to be dependent on the starting conformation, upper-rim substituent, where reduction in bulk enables faster switching, solvent and temperature with 1,2-alternate conformations switching fastest.

Heightened Dynamics of the Oxidized Y48H Variant of Human Cytochrome c Increases Its Peroxidatic Activity.

Proteins performing multiple biochemical functions are called "moonlighting proteins" or extreme multifunctional (EMF) proteins. Mitochondrial cytochrome c is an EMF protein that binds multiple partner proteins to act as a signaling molecule, transfers electrons in the respiratory chain, and acts as a peroxidase in apoptosis. Mutations in the cytochrome c gene lead to the disease thrombocytopenia, which is accompanied by enhanced apoptotic activity.

The Two-State Prehensile Tail of the Antibacterial Toxin Colicin N.

Intrinsically disordered regions within proteins are critical elements in many biomolecular interactions and signaling pathways. Antibacterial toxins of the colicin family, which could provide new antibiotic functions against resistant bacteria, contain disordered N-terminal translocation domains (T-domains) that are essential for receptor binding and the penetration of the Escherichia coli outer membrane. Here we investigate the conformational behavior of the T-domain of colicin N (ColN-T) to understand why such domains are widespread in toxins that target Gram-negative bacteria.

Near-complete backbone resonance assignments of acid-denatured human cytochrome c in dimethylsulfoxide: a prelude to studying interactions with phospholipids.

Human cytochrome c plays a central role in the mitochondrial electron transfer chain and in the intrinsic apoptosis pathway. Through the interaction with the phospholipid cardiolipin, cytochrome c triggers release of pro-apoptotic factors, including itself, from the mitochondrion into the cytosol of cells undergoing apoptosis. The cytochrome c/cardiolipin complex has been extensively studied through various spectroscopies, most recently with high-field solution and solid-state NMR spectroscopies, but there is no agreement between the various studies on key structural features of cytochrome c in its complex with cardiolipin.

Spin Saturation Transfer Difference NMR (SSTD NMR): A New Tool to Obtain Kinetic Parameters of Chemical Exchange Processes.

This detailed protocol describes the new Spin Saturation Transfer Difference Nuclear Magnetic Resonance protocol (SSTD NMR), recently developed in our group to study processes of mutual-site chemical exchange that are difficult to analyze by traditional methods. As the name suggests, this method combines the Spin Saturation Transfer method used for small molecules, with the Saturation Transfer Difference (STD) NMR method employed for the study of protein-ligand interactions, by measuring transient spin saturation transfer along increasing saturation times (build-up curves) in small organic and organometallic molecules undergoing chemical exchange. Advantages of this method over existing ones are: there is no need to reach coalescence of the exchanging signals; the method can be applied as long as one signal of the exchanging sites is isolated; there is no need to measure T1 or reach steady state saturation; rate constant values are measured directly, and T1 values are obtained in the same experiment, using only one set of experiments.

Increased dynamics in the 40-57 Ω-loop of the G41S variant of human cytochrome c promote its pro-apoptotic conformation.

Thrombocytopenia 4 is an inherited autosomal dominant thrombocytopenia, which occurs due to mutations in the human gene for cytochrome c that results in enhanced mitochondrial apoptotic activity. The Gly41Ser mutation was the first to be reported. Here we report stopped-flow kinetic studies of azide binding to human ferricytochrome c and its Gly41Ser variant, together with backbone amide H/D exchange and (15)N-relaxation dynamics using NMR spectroscopy, to show that alternative conformations are kinetically and thermodynamically more readily accessible for the Gly41Ser variant than for the wild-type protein.

Backbone resonance assignments of ferric human cytochrome c and the pro-apoptotic G41S mutant in the ferric and ferrous states.

Human cytochrome c is a multi-functional protein with key roles in both the mitochondrial electron transfer chain and in apoptosis. In the latter, a complex formed between the mitochondrial phospholipid cardiolipin and cytochrome c is crucial for instigating the release of pro-apoptotic factors, including cytochrome c, from the mitochondrion into the cytosol. The G41S mutant of human cytochrome c is the only known disease-related variant of cytochrome c and causes increased apoptotic activity in patients with autosomal dominant thrombocytopenia.

Identification and characterisation of a G-quadruplex forming sequence in the promoter region of nuclear factor (erythroid-derived 2)-like 2 (Nrf2).

The transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) regulates multiple antioxidants, Phase II detoxification enzymes and other cytoprotective enzymes in cells. Activation of Nrf2 is recognised as being of potential therapeutic benefit in inflammatory-diseases whereas more recently, it has become clear that the inhibition of Nrf2 may have benefit in the alleviation of resistance in some tumour types. A potential G-quadruplex forming sequence was identified in the promoter region of Nrf2, close to a number of putative transcription factor binding sites.

Biophysical features of bacillithiol, the glutathione surrogate of Bacillus subtilis and other firmicutes.

Bacillithiol (BSH) is the major low-molecular-weight (LMW) thiol in many low-G+C Gram-positive bacteria (Firmicutes). Evidence now emerging suggests that BSH functions as an important LMW thiol in redox regulation and xenobiotic detoxification, analogous to what is already known for glutathione and mycothiol in other microorganisms. The biophysical properties and cellular concentrations of such LMW thiols are important determinants of their biochemical efficiency both as biochemical nucleophiles and as redox buffers.

Simple computation of reaction-diffusion processes on point clouds.

The study of reaction-diffusion processes is much more complicated on general curved surfaces than on standard Cartesian coordinate spaces. Here we show how to formulate and solve systems of reaction-diffusion equations on surfaces in an extremely simple way, using only the standard Cartesian form of differential operators, and a discrete unorganized point set to represent the surface. Our method decouples surface geometry from the underlying differential operators.

Intrinsically disordered proteins: lessons from colicins.

Defining structural features of IDPs (intrinsically disordered proteins) and relating these to biological function requires characterization of their dynamical properties. In the present paper, we review what is known about the IDPs of colicins, protein antibiotics that use their IDPs to enter bacterial cells. The structurally characterized colicin IDPs we consider contain linear binding epitopes for proteins within their target cells that the colicin hijacks during entry.

Structural evidence that colicin A protein binds to a novel binding site of TolA protein in Escherichia coli periplasm.

The Tol assembly of proteins is an interacting network of proteins located in the Escherichia coli cell envelope that transduces energy and contributes to cell integrity. TolA is central to this network linking the inner and outer membranes by interactions with TolQ, TolR, TolB, and Pal. Group A colicins, such as ColA, parasitize the Tol network through interactions with TolA and/or TolB to facilitate translocation through the cell envelope to reach their cytotoxic site of action.

Characteristics and short-term prognosis of perioperative myocardial infarction in patients undergoing noncardiac surgery: a cohort study.

Background: Each year, millions of patients worldwide have a perioperative myocardial infarction (MI) after noncardiac surgery.

Objective: To examine the characteristics and short-term outcome of perioperative MI.

Design: Cohort study.

The structural and energetic basis for high selectivity in a high-affinity protein-protein interaction.

High-affinity, high-selectivity protein-protein interactions that are critical for cell survival present an evolutionary paradox: How does selectivity evolve when acquired mutations risk a lethal loss of high-affinity binding? A detailed understanding of selectivity in such complexes requires structural information on weak, noncognate complexes which can be difficult to obtain due to their transient and dynamic nature. Using NMR-based docking as a guide, we deployed a disulfide-trapping strategy on a noncognate complex between the colicin E9 endonuclease (E9 DNase) and immunity protein 2 (Im2), which is seven orders of magnitude weaker binding than the cognate femtomolar E9 DNase-Im9 interaction. The 1.

ABAM, a model for bioaccumulation of POPs in birds: validation for adult herring gulls and their eggs in Lake Ontario.

An Avian BioAccumulation Model (ABAM) of persistent organic pollutant (POP) uptake and elimination in adult life-stage of birds was validated by simulation of concentrations of DDE, dieldrin, mirex, and HCB in herring gull eggs in Lake Ontario for the years 1985, 1990, and 1992. These chemicals represented a range of whole-body half-lives of 82-265 days in the gull. Dietary intake of POPs by a female gull was simulated by a dynamic bioenergetics model which included dependence on temperature, photoperiod, egg production, and feeding chicks.

Radiocesium in caribou and reindeer in northern Canada, Alaska and Greenland from 1958 to 2000.

This study summarizes the spatial and temporal trends of fallout (137)Cs concentrations in caribou and reindeer (Rangifer tarandus ssp.), reported in various programs in Canada, Alaska and Greenland, over a 40-y period. During the 1960s, the highest (137)Cs concentrations (2000-3000Bqkg(-1) wet weight in muscle) were found in the large caribou herds of central northern Canada, with levels about 50% lower in Alaska and Greenland.

Synthesis of the branched-chain sugar aceric acid: a unique component of the pectic polysaccharide rhamnogalacturonan-II.

Described herein is the synthesis of 3-C-carboxy-5-deoxy-L-xylose (aceric acid), a rare branched-chain sugar found in the complex pectic polysaccharide rhamnogalacturonan-II. The key synthetic step in the construction of aceric acid was the stereoselective addition of 2-trimethylsilyl thiazole to 5-deoxy-1,2-O-isopropylidene-alpha-L-erythro-pentofuran-3-ulose (2), which was prepared from L-xylose. The thiazole group was efficiently converted into the required carboxyl group via conventional transformations.

Interactions of TolB with the translocation domain of colicin E9 require an extended TolB box.

The mechanism by which enzymatic E colicins such as colicin E3 (ColE3) and ColE9 cross the outer membrane, periplasm, and cytoplasmic membrane to reach the cytoplasm and thus kill Escherichia coli cells is unique in prokaryotic biology but is poorly understood. This requires an interaction between TolB in the periplasm and three essential residues, D35, S37, and W39, of a pentapeptide sequence called the TolB box located in the N-terminal translocation domain of the enzymatic E colicins. Here we used site-directed mutagenesis to demonstrate that the TolB box sequence in ColE9 is actually larger than the pentapeptide and extends from residues 34 to 46.