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Denaturation Studies (denaturation + studies)
Selected AbstractsEfficient Increase of DNA Cleavage Activity of a Diiron(III) Complex by a Conjugating Acridine GroupEUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 34 2007Xiao-Qiang Chen Abstract A new diferric complex, Fe2Lb, in which a DNA intercalator (acridine) is linked to a precursor diferric complex (Fe2La), has been designed and synthesised as a hydrolytic cleaving agent of DNA. Compared with Fe2La (without the DNA intercalator) (La: 2,6-bis{[(2-hydroxybenzyl)(pyridin-2-yl)methylamino]methyl}-4-methylphenol), Fe2Lb [Lb: 5-(acridin-9-yl)- N -(3,5-bis{[(2-hydroxybenzyl)(pyridin-2-yl)methylamino]methyl}-4-hydroxybenzyl)pentanamide] leads to a 14-fold increase in the cleavage efficiency of plasmid DNA due to the binding interaction between DNA and the acridine moiety. The interaction has been demonstrated by UV/Vis absorption, CD spectroscopy, viscidity experiments and thermal denaturation studies. The hydrolytic mechanism is supported by evidence from T4 DNA ligase assay, reactive oxygen species (ROS) quenching and BNPP [bis(4-nitrophenyl) phosphate, a DNA model] cleavage experiments. The pH dependence of the BNPP cleavage by Fe2La in aqueous buffer media shows a bell-shaped pH,kobs profile with an optimum point around a pH of 7.0 which is in good agreement with the maximum point of the pH-dependent relative concentration curve of active species from the pH titration experiments. The determination of the initial rates at a pH of 7.36 as a function of substrate concentration reveals saturation kinetics with Michaelis,Menten-like behaviour and Fe2La shows a rate acceleration increase of 4.7,×,106 times in the hydrolysis of BNPP. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007) [source] Efficient DNA Cleavage Induced by Copper(II) Complexes of Hydrolysis Derivatives of 2,4,6-Tri(2-pyridyl)-1,3,5-triazine in the Presence of Reducing AgentsEUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 6 2007Joaquín Borrás Abstract The reaction of 2,4,6-tri(pyridyl)-1,3,5-triazine (ptz) and copper(II) salts in dmf/water (1:1) results in the hydrolysis of ptz and formation of the anions bis(2-pyridylcarbonyl)amide (ptO2,) and bis(2-pyridylamine)amide (ptN2,), which are found in the complexes [Cu(ptN2)(OAc)]·3H2O (1), [Cu(ptO2)(OAc)(H2O)]·H2O (2), [Cu(ptN2)(for)]·3H2O (3) (for = formate), [Cu(ptO2)(for)(H2O)] (4), [Cu(ptO2)(benz)]·H2O (5) (benz = benzoate), and [Cu(ptO2)F(H2O)]2·3H2O (6). This report includes the chemical and spectroscopic characterization of all these complexes along with the crystal structures of 4,6. The coordination spheres of CuII in 4 and 5 are best described as distorted tetragonal square pyramidal for the former and distorted square planar for the latter. The crystal structure of 6 shows the presence of two discrete monomeric [Cu(ptO2)F(H2O)] entities in the crystallographic asymmetric unit in which both copper(II) ions have a distorted square-pyramidal coordination geometry. The binding of the complexes to DNA has been investigated with the aid of viscosity and thermal denaturation studies, both of which indicate that the interaction is probably due to the outer-sphere mechanism. The ability of the compounds to cleave DNA has also been tested. Efficient oxidative cleavage was observed in the presence of a mild reducing agent (ascorbate) and dioxygen. Mechanistic studies with reactive oxygen species (ROS) scavengers confirm that hydrogen peroxide, the hydroxyl radical, singlet oxygen-like species, and the superoxide anion are necessary diffusible intermediates in the scission process. A mechanism involving either the Fenton or theHaber,Weiss reaction plus the formation of copper oxene species is proposed for the DNA cleavage mediated by these compounds.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2007) [source] Unusual stability of human neuroglobin at low pH , molecular mechanisms and biological significanceFEBS JOURNAL, Issue 23 2009Paola Picotti Neuroglobin (Ngb) is a recently discovered globin that is predominantly expressed in the brain, retina and other nerve tissues of human and other vertebrates. Ngb has been shown to act as a neuroprotective factor, promoting neuronal survival in conditions of hypoxic,ischemic insult, such as those occurring during stroke. In this work, the conformational and functional stability of Ngb at acidic pH was analyzed, and the results were compared to those obtained with Mb. It was shown by spectroscopic and biochemical (limited proteolysis) techniques that, at pH 2.0, apoNgb is a folded and rigid protein, retaining most of the structural features that the protein displays at neutral pH. Conversely, apoMb, under the same experimental conditions of acidic pH, is essentially a random coil polypeptide. Urea-mediated denaturation studies revealed that the stability displayed by apoNgb at pH 2.0 is very similar to that of Mb at pH 7.0. Ngb also shows enhanced functional stability as compared with Mb, being capable of heme binding over a more acidic pH range than Mb. Furthermore, Ngb reversibly binds oxygen at acidic pH, with an affinity that increases as the pH is decreased. It is proposed that the acid-stable fold of Ngb depends on the particular amino acid composition of the protein polypeptide chain. The functional stability at low pH displayed by Ngb was instead shown to be related to hexacoordination of the heme group. The biological implications of the unusual acid resistance of the folding and function of Ngb are discussed. [source] Intrastrand foldamer crosslinking by reductive aminationJOURNAL OF POLYMER SCIENCE (IN TWO SECTIONS), Issue 4 2010Ronald A. Smaldone Abstract A series of m -phenylene ethynylene (mPE) foldamers were crosslinked in their helical conformation using a reductive amination-based strategy. This was accomplished by placing aldehyde moieties in the backbone of the oligomer at specific residues, which allowed a diamine crosslinker to covalently link the helical loops together. Three different foldamers with crosslinks placed at different locations in the backbone were synthesized and characterized by mass spectrometry, 1H NMR, and gel permeation chromatography. The effect of the crosslinking on the stability of the folded state was evaluated through solvent denaturation studies. These studies show a reduction in the oligomer's ability to unfold of up to 30% relative to an unmodified mPE oligomer of the same length in solvents that promote unfolding. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 927,935, 2010 [source] Integrated biophysical studies implicate partial unfolding of NBD1 of CFTR in the molecular pathogenesis of F508del cystic fibrosisPROTEIN SCIENCE, Issue 10 2010Chi Wang Abstract The lethal genetic disease cystic fibrosis is caused predominantly by in-frame deletion of phenylalanine 508 in the cystic fibrosis transmembrane conductance regulator (CFTR). F508 is located in the first nucleotide-binding domain (NBD1) of CFTR, which functions as an ATP-gated chloride channel on the cell surface. The F508del mutation blocks CFTR export to the surface due to aberrant retention in the endoplasmic reticulum. While it was assumed that F508del interferes with NBD1 folding, biophysical studies of purified NBD1 have given conflicting results concerning the mutation's influence on domain folding and stability. We have conducted isothermal (this paper) and thermal (accompanying paper) denaturation studies of human NBD1 using a variety of biophysical techniques, including simultaneous circular dichroism, intrinsic fluorescence, and static light-scattering measurements. These studies show that, in the absence of ATP, NBD1 unfolds via two sequential conformational transitions. The first, which is strongly influenced by F508del, involves partial unfolding and leads to aggregation accompanied by an increase in tryptophan fluorescence. The second, which is not significantly influenced by F508del, involves full unfolding of NBD1. Mg-ATP binding delays the first transition, thereby offsetting the effect of F508del on domain stability. Evidence suggests that the initial partial unfolding transition is partially responsible for the poor in vitro solubility of human NBD1. Second-site mutations that increase the solubility of isolated F508del-NBD1 in vitro and suppress the trafficking defect of intact F508del-CFTR in vivo also stabilize the protein against this transition, supporting the hypothesize that it is responsible for the pathological trafficking of F508del-CFTR. [source] Design, synthesis, and characterization of a novel hemoproteinPROTEIN SCIENCE, Issue 2 2001Zhijin Xu Abstract Here we describe a synthetic protein (6H7H) designed to bind four heme groups via bis,histidine axial ligation. The hemes are designed to bind perpendicular to another in an orientation that mimics the relative geometry of the two heme a groups in the active site of cytochrome c oxidase. Our newly developed protein-design program, called CORE, was implemented in the design of this novel hemoprotein. Heme titration studies resolved four distinct KD values (KD1 = 80 nM, KD2 = 18 nM, KD3 , 3 mM, KD4 , 570 nM, with KD3 × K D4 = 1700); positive cooperativity in binding between the first and second heme, as well as substantial positive cooperativity between the third and forth heme, was observed. Chemical and thermal denaturation studies reveal a stable protein with native-like properties. Visible circular dichroism spectroscopy of holo-6H7H indicates excitonic coupling between heme groups. Further electrochemical and spectroscopic characterization of the holo-protein support a structure that is consistent with the predefined target structure. [source] | ||||||||||