Thermodynamic parameters for the helix-coil thermal transition of ribonuclease-S-peptide and derivatives from 1H-NMR data

Values for the thermodynamic quantities, ΔH° = 11.8 ± 2.0 Kcal/mole and ΔS° = 43.6 ± 6.0 e.u., of the 3-13 helix–coil equilibrium of isolated S-peptide (19 residue N-terminal fragment of ribonuclease A) in aqueous solution (3 m M, 1M NaCl, pD 5.4) have been determined from a joint analysis of the Thr 3γ, Ala 6β, Phe 8meta, and Phe 8para ¹H chemical shift vs temperature curves (?7 to 80°C) in several aqueous–trifluorethanol mixtures. Chemical shifts in the coil and in the helix have been determined for up to 16 protons belonging to the 3-13 fragment. Thermodynamic parameters have also been determined for C-peptide (13 residue fragment) and a number of S-peptide derivatives. From the variation of the values of the thermodynamic parameters at pD 2.5, 5.4, and 8.0, a quantitation of the two helix-stabilizing side-chain interactions can be made: (1) Δ(ΔH°) ? 5 Kcal/mole and Δ(ΔS°) ? 18 e.u. for the salt bridge Glu 2^? … Arg 10⁺ and (2) Δ(ΔH°) ? 3 Kcal/mole and Δ(ΔS°) = 9 e.u. for the one in which the His 12⁺ imidazolium group is involved, presumably a partial stacking with the Phe 8 side chain.     

Microbial Allyl Rearrangement and Resolution of Acetates of Unsaturated Cyclic Terpene Alchols by Pseudomonas sp. NOF-5 Strain

Microbial hydrolysis of the acetates of unsaturated cyclic terpene alcohols by Pseudomonas sp. NOF-5 isolated from soil was investigated. (±)-trans-Carveyl acetate ((±)-trans-3) was enantio-selectively hydrolyzed with NOF-5 strain to give ( – )-trans-carveol (( – )-trans-2 of 86.6% optical purity). However, the hydrolysis of (±)-cis-3 was less enantioselective, while (±)-piperitylacetate ((±)-6, a cis and trans mixture) was hydrolyzed to give the ( – )-trans- and ( – )-cis-piperitols (( – )- trans-5 and ( – )-cis-5) in a poor optical yield. In this case, other tert-alcohols, ( + )-trans- and ( – )- ds-2-p-menthen-1-ols ((±)-trans-7 and ( – )-cis-7), were also produced. Furthermore, microbial and enzymic allyl rearrangements of ( + )-trans-6 and ( – )-trans-verbenylacetate (( – )-trans-11) were studied. Biological treatment of (+)-trans-6 and ( – )-trans-11 with NOF-5 or its esterase gave (+)-trans- and (-)-cis-1 and ( + )-cis-3-pinen-2-ol (( + )-cis-12), respectively.     

Inviability of a DNA2 deletion mutant is due to the DNA damage checkpoint

Dna2 is a dual polarity exo/endonuclease, and 5' to 3' DNA helicase involved in Okazaki Fragment Processing (OFP) and Double-Strand Break (DSB) Repair. In yeast, DNA2 is an essential gene, as expected for a DNA replication protein. Suppression of the lethality of dna2Δ mutants has been found to occur by two mechanisms: overexpression of RAD27^scFEN1, encoding a 5' to 3' exo/endo nuclease that processes Okazaki fragments (OFs) for ligation, or deletion of PIF1, a 5' to 3' helicase involved in mitochondrial recombination, telomerase inhibition and OFP. Mapping of a novel, spontaneously arising suppressor of dna2Δ now reveals that mutation of rad9 and double mutation of rad9 mrc1 can also suppress the lethality of dna2Δ mutants. Interaction of dna2Δ and DNA damage checkpoint mutations provides insight as to why dna2Δ is lethal but rad27Δ is not, even though evidence shows that Rad27^ScFEN1 processes most of the Okazaki fragments, while Dna2 processes only a subset.     

Thermodynamics of cyclophilin catalyzed peptidyl-prolyl isomerization by nmr spectroscopy

One-dimensional nmr exchange spectroscopy was carried out to determine thermodynamic parameters of cyclophilin-induced cis-trans isomerization of succinyl-Ala-Phe-Pro-Phe-p-nitroanilide. Rate measurements were possible at physiological temperatures. The k_c/K_m of rat cyclophilin was found to he 12.8 (±0.5) s^?1 μM^?1 at 37°C, intermediate to previously reported values that used a coupled enzyme assay extrapolated to this temperature. Activation energies (ΔG^≠) for the uncatalyzed and catalyzed reaction at 37°C were found to be 19.7 and 17.1 kcal/mol, respectively, and were primarily due to an enthalpic barrier. © 1994 John Wiley & Sons, Inc.     

Nonionic but water soluble, [Glycine-Pd-Alanine] and [Glycine-Pd-Valine] complexes. Their synthesis,characterization, antitumor activities and rich DNA/HSA interaction studies

Two novel, neutral and water soluble Pd(II) complexes of formula [Pd(Gly)(Ala)] (1) and [Pd(Gly)(Val)] (2) (Gly, Ala, and Val are anionic forms of glycine, alanine, and valine amino acids, respectively) have been synthesized and characterized by FT-IR, UV–Vis, ¹H-NMR, elemental analysis, and molar conductivity measurement. The data revealed that each amino acid binds to Pd(II) through the nitrogen of –NH₂ and the oxygen of –COO^– groups and acts as a bidentate chelate. These complexes have been assayed against leukemia cells (K₅₆₂) using MTT method. The results indicated that both of the complexes display more cytotoxicity than the well-known anticancer drug, cisplatin. The interaction of the compounds with calf thymus DNA (CT-DNA) and human serum albumin (HSA) were assayed by a series of experimental techniques including electronic absorption, fluorescence, viscometry, gel electrophoresis, and FT-IR. The results indicated that the two complexes have interesting binding propensities toward CT-DNA as well as HSA and the binding affinity of (1) is more than (2). The fluorescence data indicated that both complexes strongly quench the fluorescence of ethidium bromide–DNA system as well as the intrinsic fluorescence of HSA via static quenching procedures. The thermodynamic parameters (ΔH°, ΔS°, and ΔG°) calculated from the fluorescence studies showed that hydrogen bonds and van der Waals interactions play a major role in the binding of the complexes to DNA and HSA. We suggest that both of the Pd(II) complexes exhibit the groove binding mode with CT-DNA and interact with the main binding pocket of HSA.

Communicated by Ramaswamy H. Sarma     

Adsorption Optimization of Lead (II) Using Saccharum Bengalense as a Non-Conventional Low Cost Biosorbent: Isotherm and Thermodynamics Modeling

In the present study a novel biomass, derived from the pulp of Saccharum bengalense, was used as an adsorbent material for the removal of Pb (II) ions from aqueous solution. After 50 minutes contact time, almost 92% lead removal was possible at pH 6.0 under batch test conditions. The experimental data was analyzed using Langmuir, Freundlich, Timken and Dubinin-Radushkevich two parameters isotherm model, three parameters Redlich—Peterson, Sip and Toth models and four parameters Fritz Schlunder isotherm models. Langmuir, Redlich—Peterson and Fritz-Schlunder models were found to be the best fit models. Kinetic studies revealed that the sorption process was well explained with pseudo second-order kinetic model. Thermodynamic parameters including free energy change (ΔG°), enthalpy change (ΔH°) and entropy change (ΔS°) have been calculated and reveal the spontaneous, endothermic and feasible nature of the adsorption process. The thermodynamic parameters of activation (ΔG ^#, ΔH ^#and ΔS ^#) were calculated from the pseudo-second order rate constant by using the Eyring equation. Results showed that Pb (II) adsorption onto SB is an associated mechanism and the reorientation step is entropy controlled.     

Salt effects on the denaturation of DNA. V. Preferential interactions of native and denatured calf thymus DNA in Na2SO4 solutions of varying ionic strength.

Precision density measurements were performed at 25°C on Na-DNA-Na₂SO₄ mixtures in the presence of either 0.005 m cacodylic acid buffer (pH 6.8) or in the presence of 0.1 m NaOH (pH 12.3). From measurements executed under equilibrium dialysis conditions, the so-called “density increments” (?ρ/?c₂)_μ⁰ for native (pH 6.8), heat-denatured (pH 6.8), and alkali-denatured (pH 12.6) Na-DNA were evaluated as a function of Na₂SO₄ concentration. (?ρ/?c₂)_μ⁰ for native DNA was found to decrease almost linearly with ionic strength I^1/2 of the supporting electrolyte. The density increment for Na-DNA at pH 12.6, on the other hand, increases in more or less linear fashion with I^1/2. (?ρ/?c₂)_μ⁰ for heat-denatured DNA at pH 6.8 is not affected very much by increasing salt strength. From density measurements performed on the Na-DNA–Na₂SO₄ mixtures at fixed concentrations of diffusible components, the partial specific volumes ν ₂° of native (pH 6.8), heat-denatured (pH 6.8), and alkali-denatured (pH 12.6) Na-DNA were determined as a function of Na₂SO₄ concentration. All ν ₂° values, irrespective of the secondary structure of the DNA, increase with increasing salt concentration although the increase for heat denatured DNA (pH 6.8) is barely noticeable. ν ₂° of both native and heat-denatured DNA (pH 6.8) extrapolates to a value of 0.50_o ml/g at vanishing salt concentration; ν ₂° of DNA in 0.1 m NaOH, on the other hand, assumes the value 0.2_o ml/g. Distribution coefficients of diffusible components, expressed in terms of preferential water and salt interaction, were evaluated from the (?ρ/?c₂)_μ⁰ data, solvent densities, and partial specific volumes of all solution components. All interaction parameters depend strongly on salt concentration and on the conformation of DNA. From data collected and from information available in the literature it is concluded that Na₂SO₄, for instance, displaces water of hydration from native DNA much more readily with increasing salt concentration than does NaCl. The solvation properties of the denatured forms of Na-DNA are rather complex but appear to be in harmony with whatever information can be gathered from the literature.     

Genetic manipulation of a latent defect in yeast cytochrome biosynthesis utilizing cytoduction.

The effects of a mit^? mutation, oxi2, and the ρ° mutation on expression of a defective nuclear structural gene for δ-aminolevulinic acid synthase (cyd1) were compared. The technique of cytoduction was used to introduce oxi2 mitochondria into a cyd1ρ° recipient cell, thereby permitting comparison of isonuclear strains. Like ρ°, the oxi2 mutation caused an apparent unmasking of the cytochrome deficiency associated with the cyd1 mutation, provided cells were grown on glucose. When cyd1 strains with ρ⁺, ρ° or oxi2 mitochondrial genotypes were grown on galactose medium, substantial cytochrome formation occurred in each case. It is concluded that the exacerbation of the cyd1-dependent cytochrome deficiency by ρ° or oxi2 mutations depends upon glucose repression. However, derepression resulting from growth on galactose medium does not fully overcome the cyd1 defect, since both cyd1ρ°oxi2 strains require added δ-aminolevulinic acid for maximum cytochrome biosynthesis.     

Electronic and fluorescent studies on the interaction of DNA and BSA with a new ternary praseodymium complex containing 2,9-dimethyl 1,10-phenanthroline and antibacterial activities testing

In this study, fluorescence emission spectra, UV–vis absorption spectra, ethidium bromide (EB)-competition experiment, and iodide quenching experiment were used for the interaction study of the Fish salmon DNA (FS-DNA) with [Pr(dmp)2Cl3(OH2)] where dmp is 2,9-dimethyl 1,10-phenanthroline. The binding constant and the number of binding sites of the complex with FS-DNA were 6.09?±?0.04 M^?1 and 1.18, respectively. The free energy, enthalpy, and entropy changes (ΔG°, ΔH°, and ΔS°) in the binding process of the Pr(III) complex with FS-DNA were –8.02?kcal mol^?1, +39.44?kcal mol^?1, and +159.56?cal mol^?1 K^?1, respectively. Based on these results, the interaction process between FS-DNA with [Pr(dmp)2Cl3(OH2)] was spontaneous and the main binding interaction force was groove binding mode. Also, Fluorescence and electronic absorption spectroscopy were used in order to evaluate the binding characteristics, stoichiometry, and interaction mode of praseodymium(III) (Pr(III)) complex with bovine serum albumin (BSA). Title complex showed good binding propensity to BSA presenting moderately high Kb values. The fluorescence quenching of BSA by Pr(III) complex has been observed to be the static process. The positive ΔH° and ΔS° values showed that the hydrophobic interaction is the main force in the binding of Pr(III) complex and BSA. Eventually, the average aggregation number, <J>, of BSA potentially induced by title complex confirmed the 1:1 stoichiometry for title complex-BSA adducts. In vitro, antimicrobial activity of title complex was indicated that the complex is more active against both Escherichia coli and Enterococcus faecalis bacterial strains than Staphylococcus aureus, and Pseudomonas aeruginosa.

Communicated by Ramaswamy H. Sarma     

The random coil → β transition of copolymers of L-lysine and L-isoleucine: Potentiometric titration and circular dichroism studies

Copolymers of L -lysine and L -isoleucine [poly(L -Lys^f,L -Val^{1 ? f})] containing 4–15% isoleucine were investigated using potentiometric titration and circular dichroism (CD) spectroscopy. With increasing isoleucine content, β-sheet formation is favored over α-helix formation at high pH and room temperature. The fraction of β-sheet present, as a function of pH, calculated from titrations of poly(L -Lys^85.2,L -Ile^14.8), agreed well with data obtained from CD studies for the same copolymer. Thermodynamic parameters were determined from titrations using the method of Zimm and Rice; the partial free energy (ΔG°_{C → β}) at 25° for the coil-to-β-sheet transition for isoleucine was estimated to be ?515 cal/mol; from the temperature dependence of free energy, the partial entropy (ΔS°_cβ), and the partial free enthalpy (ΔH°_{c → β}) of the coil → β transition for isoleucine is estimated to be 2.6 e.u. and 260 cal/mol, respectively. The partial thermodynamic parameters obtained for lysine are in good agreement with literature values. It is concluded from these studies that isoleucine has a very high potential for a β-sheet formation.     

Kinetics and thermodynamics of catalysis and thermal inactivation of a novel α-amylase (Tp-AmyS) from Thermotoga petrophila

Abstract

This research is focussed on kinetic, thermodynamic and thermal inactivation of a novel thermostable recombinant α-amylase (Tp-AmyS) from Thermotoga petrophila. The amylase gene was cloned in pHIS-parallel1 expression vector and overexpressed in Escherichia coli. The steady-state kinetic parameters (V_max, K_m, k_cat and k_cat/K_m) for the hydrolysis of amylose (1.39?mg/min, 0.57?mg, 148.6?s^?1, 260.7), amylopectin (2.3?mg/min, 1.09?mg, 247.1?s^?1, 226.7), soluble starch (2.67?mg/min, 2.98?mg, 284.2?s^?1, 95.4) and raw starch (2.1?mg/min, 3.6?mg, 224.7?s^?1, 61.9) were determined. The activation energy (E_a), free energy (ΔG), enthalpy (ΔH) and entropy of activation (ΔS) at 98?°C were 42.9?kJ mol^?1, 74?kJ mol^?1, 39.9?kJ mol^?1 and ?92.3 J mol^?1 K^?1, respectively, for soluble starch hydrolysis. While ΔG of substrate binding (ΔG_E-S) and ΔG of transition state binding (ΔG_E-T) were 3.38 and ?14.1?kJ mol^?1, respectively. Whereas, EaD, Gibbs free energy (ΔG*), increase in the enthalpy (ΔH*) and activation entropy (ΔS*) for activation of the unfolding of transition state were 108, 107, 105?kJ mol^?1 and ?4.1 J mol^?1 K^?1. The thermodynamics of irreversible thermal inactivation of Tp-AmyS revealed that at high temperature the process involves the aggregation of the protein.     

Upregulation of P-glycoprotein in rat hepatoma ρ° cells: Implications for drug–DNA interactions

Rat hepatoma cells lacking mitochondrial DNA (ρ° cells) were used as a model system to examine the possible roles of mitochondrial DNA as a target for the DNA-acting anticancer drug Adriamycin (doxorubicin). The ρ° cells were 45-fold less sensitive to Adriamycin than the parental ρ⁺ cells containing mitochondrial DNA. Other non-DNA-acting drugs also exhibited similar behaviour, and this was shown to be due to a multidrug resistance (MDR) phenotype in the ρ° cells. This was indicated by confocal microscopy where ρ⁺ cells exhibited thirteenfold higher cellular levels of Adriamycin than ρ° cells. Upregulation (tenfold) of P-glycoprotein in ρ° cells was also confirmed by Northern dot blot analysis. Since the MDR phenotype is present in ρ° cells and upregulation of P-glycoprotein is maintained in these cells, ρ° cells are not a good model system for drug-DNA studies (where the drug is susceptible to extrusion by P-glycoprotein), and any such results obtained with this system must be treated with considerable caution. J. Cell. Biochem. 69:463–469, 1998. © 1998 Wiley-Liss, Inc.     

Multiple interconverting conformers of the cyclic tetrapeptide tentoxin, [cyclo-(L-MeAla1-L-Leu2-MePhe[(Z)Δ]3-Gly4)], as seen by two-dimensional 1H-nmr spectroscopy

The conformations of the phytotoxic cyclic tetrapeptide tentoxin [cyclo-(L -MeAla¹-L -Leu²-MePhe[(Z)Δ]³-Gly⁴ )] have been studied in aqueous solution by two-dimensional proton nmr at various temperatures. Contrary to what is observed in chloroform, tentoxin exhibits multiple exchanging conformations in water. Aggregation phenomena were also observed. Four conformations with different proportions (51, 37, 8, and 4%) were observed at ?5°C. Models were constructed from nmr parameters and restrained molecular dynamics simulations. All the models exhibit cis-trans-cis-trans conformation of the amide bond sequence. The conversion from one form to another is accomplished by a conformational peptide flip consisting of a 180° rotation of a nonmethylated peptide bond. © 1995 John Wiley & Sons, Inc.     

A Theoretical Model for the Binding of Cis-Pt(NH3)2 +2 to DNA

Abstract

The binding of cis-Pt(NH₃)₂B₁B2 to the bases B₁ and B₂, i.e., guanine (G), cytosine (C), adenine (A), and thymine (T), of DNA is studied theoretically. The components of the binding are analyzed and a model structure is proposed for the intrastrand binding to the dB,pdB₂ sequence of a kinked double helical DNA. Quantum mechanical calculations of the ligand binding energy indicates that cw-Pt(NH₃)₂ ⁺² (cis-PDA) binds to N7(G), N3(C), 02(C), 06(G), N3(A), N7(A), 04(T) and 02(T) in order of decreasing binding energy. Conformational analysis provides structures of kinked DNA in which adjacent bases chelate to cis-PDA. Only bending toward the major groove allows the construction of acceptable square planar complexes. Examples are presented for kinks of ?70° and ?40° at the receptor site to orient the base pairs for ligand binding to B, and B₂ to form a nearly square planar complex. The energies for complex formation of cis-PDA to the various intra-strand base sites in double stranded DNA are estimated. At least 32 kcal/mole separates the energetically favorable dGpdG·cis-PDA chelate from the dCpdG·cis-PDA chelate. All other possible chelate structures are much higher in energy which correlates with their lack of observation in competition with the preferred dGpdG chelate.

The second most favorable ligand energy occurs with N3(C). A novel binding site involving dC(N3)pdG(N7) is examined. Denaturation can result in an anti ? syn rotation of C about its glycosidic bond to place N3(C) in the major groove for intrastrand binding in duplex DNA. This novel intrastrand dCpdG complex and the most favored dGpdG structure are illustrated with stereographic projections.     

A yeast protein analogous to Escherichia coli RecA protein whose cellular level is enhanced after UV irradiation

Summary In Saccharomyces cerevisiae, a protein was recognized by polyclonal antibodies raised against homogeneous Escherichia coli K12 RecA protein. The cellular level of the yeast protein called RecAsc (molecular weight 44 kDa, pI 6.3), was transiently enhanced after UV irradiation. Protease inhibitors were required to minimize degradation of the RecAsc protein during cell lysis. The RecAsc protein exhibited similar basal levels and similar kinetics of increase after UV irradiation in DNA-repair proficient (RAD ⁺) strains carrying mitochondrial DNA or not (rho ⁰). This was also true for the following DNA-repair deficient (rad ^-) strains: rad2-6 rad6-1 rad52-1, a triple mutant blocked in three major repair pathways; rad6-, a mutant containing an integrative deletion in a gene playing a central role in mutagenesis; pso2-1, a mutant that exhibits a reduced rate of mutagenesis and recombination after exposure to DNA cross-linking agents.     

Binding affinity of pyrano[3, 2-f]quinoline and DNA: spectroscopic and docking approach

The interaction between pyrano[3, 2-f]quinoline (PQ) and calf thymus DNA (CTDNA) using spectroscopic and molecular modeling approach has been presented here. Apparent association constant (1.05×10⁵ L/mol) calculated from UV-vis specta, indicates a moderate complex formation between CTDNA and PQ. The quenching phenomena as obtained from emission spectra of ethidium bromide (EB)–CTDNA by PQ was found to be a dynamic one and the binding constants found to be 8.64, 9.25, 11.17, 12.03 × 10⁴ L/mol at 293, 300, 308, and 315 K. Thermodynamic parameter enthalpy change (ΔH) and entropy change (ΔS), indicates weak force like van der Walls force and hydrogen bonds having the key role in this binding process. The results of circular dichroism (CD) demonstrate that PQ has not induced characteristic changed in CTDNA. Results achieved from UV absorption and fluorescence spectroscopy indicating the binding mode of PQ with DNA seems to be a nonintercalative binding. The theoretical results as originating from molecular modeling showed that PQ possibly will bind into the hydrophobic region of DNA having docking binding energy = ?10.03 kcal/mol and the obtained results are in consonance with the inferences obtained from experimental data. This result is important for the better understanding of pharmaceutical aspects of binding affinity of PQ and CTDNA.     

Fluorescent bovine serum albumin interacting with the antitussive quencher dextromethorphan: a spectroscopic insight

The interaction of dextromethorphan hydrobromide (DXM) with bovine serum albumin (BSA) is studied by using fluorescence spectra, UV–vis absorption, synchronous fluorescence spectra (SFS), 3D fluorescence spectra, Fourier transform infrared (FTIR) spectroscopy and circular dichroism under simulated physiological conditions. DXM effectively quenched the intrinsic fluorescence of BSA. Values of the binding constant, K_A, are 7.159 × 10³, 9.398 × 10³ and 16.101 × 10³ L/mol; the number of binding sites, n, and the corresponding thermodynamic parameters ΔG°, ΔH° and ΔS° between DXM and BSA were calculated at different temperatures. The interaction between DXM and BSA occurs through dynamic quenching and the effect of DXM on the conformation of BSA was analyzed using SFS. The average binding distance, r, between the donor (BSA) and acceptor (DXM) was determined based on Förster's theory. The results of fluorescence spectra, UV–vis absorption spectra and SFS show that the secondary structure of the protein has been changed in the presence of DXM. Copyright © 2015 John Wiley & Sons, Ltd.