Photo-induced double-strand DNA and site-specific protein cleavage activity of L-histidine (mu-oxo)diiron(III) complexes of heterocyclic bases

Three oxo-bridged diiron(III) complexes of L-histidine and heterocyclic bases [Fe-2(mu-O)(L-his)(2)(B)(2)](ClO4)(2) (1-3), where B is 2,2'-bipyridine (bpy),1,10-phenanthroline (phen), dipyrido[3,2-d:2',3'-f]quinoxaline (dpq), were prepared and characterized. The bpy complex 1 was structurally characterized by X-ray crystallography. The molecular structure showed a {Fe-2(mu-O)} core in which iron(III) in a FeN4O2 coordination is bound to tridentate monoanionic L-histidine and bidentate bpy ligands. The Fe center dot center dot center dot Fe distance is similar to 3.5 angstrom. The Fe-O-Fe unit is essentially linear, giving a bond angle of similar to 172 degrees. The complexes showed irreversible cyclic voltammetric cathodic response near -0.1 V vs. SCE in H2O-0.1 M KCl. The binuclear units displayed antiferromagnetic interaction between two high-spin (S = 5/2) iron(III) centers giving a -J value of -110 cm(-1). The complexes showed good DNA binding propensity giving a binding constant value of similar to 10(5) M-1. Isothermal titration calorimetric data indicated single binding mode to the DNA. The binding was found to be driven by negative free energy change and enthalpy. The dpq complex 3 showed oxidative double-strand DNA cleavage on exposure to UV-A and visible light. The phen complex 2 displayed single-strand photocleavage of DNA. The DNA double-strand breaks were rationalized from theoretical molecular docking calculations. Mechanistic investigations showed formation of hydroxyl radicals as the reactive species through photodecarboxylation of the L-histidine ligand. The complexes exhibited good binding propensity to bovine serum albumin (BSA) protein in Tris-HCl/NaCl buffer medium. The dpq complex 3 showed UV-A light-induced site-specific oxidative BSA cleavage forming fragments of similar to 45 kDa and similar to 20 kDa molecular weights via SOH pathway.

Small local variations in B-form DNA lead to a large variety of global geometries which can accommodate most DNA-binding protein motifs

An important question of biological relevance is the polymorphism of the double-helical DNA structure in its free form, and the changes that it undergoes upon protein-binding. We have analysed a database of free DNA crystal structures to assess the inherent variability of the free DNA structure and have compared it with a database of protein-bound DNA crystal structures to ascertain the protein-induced variations.

Effect of Crowding Agents, Signal Peptide, and Chaperone SecB on the Folding and Aggregation of E. coli Maltose Binding Protein

SecB, a soluble cytosolic chaperone component of the Secexport pathway, binds to newly synthesized precursor proteins and prevents their premature aggregation and folding and subsequently targets them to the translocation machinery on the membrane. PreMBP, the precursor form of maltose binding protein, has a 26-residue signal sequence attached to the N-terminus of MBP and is a physiological substrate of SecB. We examine the effect of macromolecular crowding and SecB on the stability and refolding of denatured preMBP and MBP. PreMBP was less stable than MBP (ΔTm =7( 0.5 K) in both crowded and uncrowded solutions. Crowding did not cause any substantial changes in the thermal stability ofMBP(ΔTm=1(0.4 K) or preMBP (ΔTm=0(0.6 K), as observed in spectroscopically monitored thermal unfolding experiments. However, both MBP and preMBP were prone to aggregation while refolding under crowded conditions. In contrast to MBP aggregates, which were amorphous, preMBP aggregates form amyloid fibrils.Under uncrowded conditions, a molar excess of SecB was able to completely prevent aggregation and promote disaggregation of preformed aggregates of MBP. When a complex of the denatured protein and SecB was preformed, SecB could completely prevent aggregation and promote folding of MBP and preMBP even in crowded solution. Thus, in addition to maintaining substrates in an unfolded, export-competent conformation, SecB also suppresses the aggregation of its substrates in the crowded intracellular environment. SecB is also able to promote passive disaggregation of macroscopic aggregates of MBP in the absence of an energy source such as ATP or additional cofactors. These experiments also demonstrate that signal peptide can reatly influence protein stability and aggregation propensity.

Minority-Carrier Lifetime In Polycrystalline Semiconductors

A model is described for grain boundary recombination in polycrystalline semiconductors. This model enables the evaluation of minority carrier lifetime in these materials. Es vvird ein Modell fur die Korngrenzenrekombination in polykristallinen Halbleitern beschrieben. Das Modell ermoglicht die Bestimmung der Minoritiitsladungstragerlebensdauer in diesen Materialien.

Kinetics of interaction of Cotton Leaf Curl Kokhran Virus-Dabawali (CLCuKV-Dab) coat protein and its mutants with ssDNA

Gemini viral assembly and transport of viral DNA into nucleus for replication, ssentially involve DNA-coat protein interactions. The kinetics of interaction of Cotton LeafCtirl Kokhran Virus-Dabawali recombinant coat protein (rCP) with DNA was studied by electrophoretic mobility shift assay (EMSA) and Surface plasmon resonance (SPR). The rCP interacted with ssDNA with a K-A, of 2.6 +/- 0.29 x 10(8) M-1 in a sequence non-specific manner. The CP has a conserved C2H2 type zinc finger motif composed of residues C68, C72, H81 and H85. Mutation of these residues to alanine resulted in reduced binding to DNA probes. The H85A mutant rCP showed the least binding with approximately 756 fold loss in the association rate and a three order magnitude decrease in the binding affinity as compared to rCP. The CP-DNA interactions via the zinc finger motif could play a Crucial role ill Virus assembly and in nuclear transport. (C) 2009 Elsevier Inc.

Nonlinear transport in semiconducting polymers at high carrier densities

Conducting and semiconducting polymers are important materials in the development of printed, flexible, large-area electronics such as flat-panel displays and photovoltaic cells. There has been rapid progress in developing conjugated polymers with high transport mobility required for high-performance field-effect transistors (FETs), beginning(1) with mobilities around 10(-4) cm(2) V-1 s(-1) to a recent report(2) of 1 cm(2) V-1 s(-1) for poly(2,5-bis(3-tetradecylthiophen-2-yl) thieno[3,2-b] thiophene) (PBTTT). Here, the electrical properties of PBTTT are studied at high charge densities both as the semiconductor layer in FETs and in electrochemically doped films to determine the transport mechanism. We show that data obtained using a wide range of parameters (temperature, gate-induced carrier density, source-drain voltage and doping level) scale onto the universal curve predicted for transport in the Luttinger liquid description of the one-dimensional `metal'.

Future switching technology: bacterial protein-based programmable all-optical switch for integrated optics applications?

We report on the bacterial protein-based all-optical switches which operate at low laser power, high speed and fulfil most of the requirements to be an ideal all-optical switch without any moving parts involved. This consists of conventional optical waveguides coated with bacteriorhodopsin films at switching locations. The principle of operation of the switch is based on the light-induced refractive index change of bacteriorhodopsin. This approach opens the possibility of realizing proteinbased all-optical switches for communication network, integrated optics and optical computers.

Purification and properties of riboflavin-binding protein from the egg white of the duck (Anas platyrhynckos).

Riboflavin-binding protein was purified from the egg white of domestic duck and some of its properties were investigated. The protein was homogeneous by the criteria of gel filtration on Sephadex G-100 and electrophoresis on sodium dodecyl sulphate-polyacrylamide gels, had molecular weight of 36 000 ± 1000 and, unlike the chicken egg white protein (Mr 32 000 ± 2000), was devoid of covalently-bound carbohydrate. It was similar to the chicken riboflavin-binding protein in its behavior on ion-exchange celluloses and affinity to interact with the flavin and its coenzymes, but differed significantly in amino acid composition in that it completely lacked proline and contained less of methionine and arginine. The protein partially cross-reacted with the specific antiserum to chicken riboflavin-binding protein with a spur during immunodiffusion analysis.

Frequency and temperature-dependent mobility of a charged carrier on a randomly interrupted strand

A randomly interrupted strand model of a one-dimensional conductor is considered. An exact analytical expression is obtained for the temperature-dependent ac mobility for a finite segment drawn at random, taking into account the reflecting barriers at the two open ends. The real part of mobility shows a broad resonance as a function of both frequency and temperature, and vanishes quadratically in the dc limit. The frequency (temperature) maximum shifts to higher values for higher temperatures (frequencies).

Nucleosome core protein: Asymmetric dissociation of the octamer

The octameric nucleosomal core-histone complex, (H2A)2-(H2B)2-(H3)2-(H4)2, isolated from rat liver, undergoes dissociation during gel exclusion chromatography as a result of dilution occurring in the columns. The elution pattern at pH 7.0 and 4°C showed a sharp leading peak containing all four histones but predominantly H3 and H4, and a trailing peak containing equal amounts of histones H2A and H2B. As column length was increased the area under the leading peak decreased and that under the trailing peak increased. In addition the relative positions of the two peaks varied with column length. From an analysis of the data on increase in elution volume of the leading peak in relation to column length an apparent molecular weight of 86 000 was calculated for the undissociated molecule. Its apparent molecular weight, histone composition and pattern of further dissociation in relation to column length suggest that this species is the hexamer, (H2A-H2B)-(H3)2-(H4)2. At pH 7.0 and 4°C the dissociation of the core complex appears to be as follows: (H2A)2-(H2B)2-(H3)2-(H4)2 → (H2A-H2B) + (H2A-H2B)-(H3)2-(H4)2 → 2(H2A-H2B) + (H3)2-(H4)2 This dissociation was accelerated by an increase in temperature or decrease in pH and was accompanied by marked conformational changes as judged by circular dichroism measurements.

Interaction of rat testis protein, TP, with nucleic acids in vitro. Fluorescence quenching, UV absorption, and thermal denaturation studies

The nucleic acid binding properties of the testis protein, TP, were studied with the help of physical techniques, namely, fluorescence quenching, UV difference absorption spectroscopy, and thermal melting. Results of quenching of tyrosine fluorescence of TP upon its binding to double-stranded and denatured rat liver nucleosome core DNA and poly(rA) suggest that the tyrosine residues of TP interact/intercalate with the bases of these nucleic acids. From the fluorescence quenching data, obtained at 50 mM NaCl concentration, the apparent association constants for binding of TP to native and denatured DNA and poly(rA) were calculated to be 4.4 X 10(3) M-1, 2.86 X 10(4) M-1, and 8.5 X 10(4) M-1, respectively. UV difference absorption spectra upon TP binding to poly(rA) and rat liver core DNA showed a TP-induced hyperchromicity at 260 nm which is suggestive of local melting of poly(rA) and DNA. The results from thermal melting studies of binding of TP to calf thymus DNA at 1 mM NaCl as well as 50 mM NaCl showed that although at 1 mM NaCl TP brings about a slight stabilization of the DNA against thermal melting, a destabilization of the DNA was observed at 50 mM NaCl. From these results it is concluded that TP, having a higher affinity for single-stranded nucleic acids, destabilizes double- stranded DNA, thus behaving like a DNA-melting protein.

Estrogen Induction Of Biotin-Binding Protein In Immature Chicks - Kinetics, Hormonal Specificity And Modulation

Employing a specific radioimmunoassay for quantification, the kinetics of estrogen-induced elevation in the plasma concentration of biotin-binding protein (BBP) in immature male chicks was investigated. A single injection of the steroid hormone enhanced the plasma BBP content several-fold at 6 h, reaching peak levels around 48 h and declining thereafter. A 2-fold amplification of the response was evident during secondary stimulation with the hormone. The magnitude of the response was hormonal dose-dependent while the initial lag phase and the time of peak protein accumulation were unaltered within the hormonal doses tested. The circulatory half-life of the specific protein in normal and estrogenized birds was 10 h. Hyperthyroidism markedly decreased the hormonal response while the opposite effect was seen during hypothyroidism. The antiestrogens E- and Z-clomiphene citrate effectively blocked the protein induction whereas progesterone, either alone or in combination with estrogen, was ineffective in modulating the induction. Cycloheximide administration drastically inhibited the inductive response. The above observations clearly suggest that the genes corresponding to the two isofunctional proteins of chicken egg, viz. BBP and avidin, are differentially regulated.

Baker's yeast expressing the Japanese encephalitis virus envelope protein on its cell surface: induction of an antigen-specific but non-neutralizing antibody response

Live recombinant Saccharomyces cerevisiae yeast expressing the envelope antigen of Japanese encephalitis virus (JEV) on the outer mannoprotein layer of the cell wall were examined for their ability to induce antigen-specific antibody responses in mice. When used as a modelantigen, parenteral immunization of mice with surface-expressing GFP yeast induced a strong anti-GFP antibody response in the absence of adjuvants. This antigen delivery approach was then used for a more stringent system, such as the envelope protein of JEV, which is a neurotropic virus requiring neutralizing antibodies for protection.Although 70% of cells were detected to express the total envelope protein on the surface by antibodies raised to the bacterially expressed protein, polyclonal anti-JEV antibodies failed to react with them. In marked contrast, yeast expressing the envelope fragments 238-398, 373-399 and 373-500 in front of a Gly-Ser linker were detected by anti-JEV antibodies as well as a monoclonal antibody but not by antibodies raised to the bacterially expressed protein. Immunization of mice with these surface-expressing recombinants resulted in a strong antibody response. However, the antibodies failed to neutralize the virus, although the fragments were selected based on neutralizing determinants.

p53 and little brother p53/47: linking IRES activities with protein functions

The tumor suppressor p53 represents a paradigm for gene regulation. Its rapid induction in response to DNA damage conditions has been attributed to both increased half-life of p53 protein and also increased translation of p53 mRNA. Recent advances in our understanding of the post-transcriptional regulation of p53 include the discovery of internal ribosome entry sites (IRESs) within the p53 mRNA. These IRES elements regulate the translation of the full length as well as the N-terminally truncated isoform, p53/47. The p53/47 isoform is generated by alternative initiation at an internal AUG codon present within the p53 ORF. The aim of this review is to summarize the role of translational control mechanisms in regulating p53 functions. We discuss here in detail how diverse cellular stress pathways trigger alterations in the cap-dependent and cap-independent translation of p53 mRNA and how changes in the relative expression levels of p53 isoforms result in more differentiated p53 activity.

Isolation and characterization of riboflavin-binding protein from pregnant-rat serum

A high-affinity riboflavin -binding protein was isolated and characterized for the first time from pregnant-rat sera by affinity chromatography on a lumiflavin-agarose column. The purified protein was homogeneous by the criteria of analytical polyacrylamide-gel disc electrophoresis, gel-filtration chromatography on Sephadex G-100 and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. It had a molecular weight of 90000+/-5000 and interacted with [14C]riboflavin with a 1:1 molar ratio with a dissociation constant (Kd) of 0.42 micron.