Infrared and X-ray diffraction study of the effect of protonation of DNA on its B-to-A transition

The influence of H+ on the secondary structure of DNA and on its B-to-A transition has been studied by employing X-ray diffraction and infrared spectroscopy. Helical parameters for DNA molecules with different degrees of protonation were determined. It was shown that H+ binding stabilizes the B-form of DNA in fibers over a wide range of water and inorganic salt content. Only 0.03 H+ bound per nucleotide is sufficient to prevent the B-to-A transition caused by decreasing relative humidity in DNA fibers containing 4% NaCl. The effectiveness of B-form stabilization by H+ is explained by changes in DNA-solvent molecule interactions, especially in the major groove of double helices.     

Influence of Ca2+ cations on low pH-induced DNA structural transitions

A confocal Raman microspectrometer was used to investigate the influence of Ca2+ cations on low pH-induced DNA structural changes. The effects of Ca2+ cations on the protonation mechanism of opening AT and changing the protonation of GC base pairs in DNA are discussed. Based on the observation that the midpoint of the transition of Watson-Crick GC base pairs to protonated GC base pairs lies at around pH 3 (analyzing the 681 cm(-1) line), measurements were carried out on calf thymus DNA at neutral pH and pH 3 in the presence of low and high concentrations of Ca2+ cations. Raman spectra show that low concentrations of Ca2+ cations partially protect DNA against protonation of cytosine (characteristic line at 1262 cm(-1)) and do not protect adenine (characteristic line at 1304 cm(-1)) and the N(7) of guanine (line at 1488 cm(-1)) against binding of H+. High Ca2+ concentrations can prevent protonation of cytosine and protonation of adenine (little disruption of AT pairs). Analyzing the line at 1488 cm(-1), which obtains most of its intensity from a guanine vibration, high salt was also found to protect the N(7) of guanine against protonation.     

The effect of hydrogen ions on the B-A transition in DNA

The effects of hydrogen ions binding to DNA on its secondary structure and B to A transition were studied by methods of X-ray diffraction and infrared spectroscopy. Helical parameters of DNA molecules with different degrees of protonation were determined. It was shown that H+-ions binding stabilize the B form of DNA in fibers in the wide range of water and inorganic salt content. Only 0.03 H+-ions bound to each nucleotide are sufficient to prevent B to A transition caused by a relative humidity decrease in DNA fibers, containing 4% of NaCl. The effective stabilization of the DNA B form by H+-ions binding is explained by modifications in DNA - solvent molecules interactions, especially in the major groove of double helices.     

Interaction of immobilized DNA with silver ions

The interaction of Ag+ with DNA immobilized in polyacrylamide gel was studied by means of the ion-exchange method. Ag+ ions are shown to bind to DNA bases, their charges being neutralized by phosphate groups. The binding sites of Ag+ and H+ are likely to be the same, but the strength of Ag+ binding is greater than that of H+. Ag+ ions like H+ are shown to cause the formation of compact structures in immobilized DNA, the amount of these structures being dependent on subtle differences in DNA samples. DNA samples, not forming compact structures under the influence of H+, do not form them under the influence of Ag+. This fact can indicate the similarity of the mechanisms of the compact structures formation in both cases. The results obtained are compared with the data available for the interaction of Ag+ with DNA in solution. The mechanism of the Ag+-DNA interaction is discussed.     

A compact form of DNA in solution. III. Influence of the ion composition of the solution on the compactization process of double-stranded DNA in the presence of peg]

The data showing the features of the DNA compactization process in PEG-containing solutions of chlorides of different alkaline metals (LiCl, KCl, RbCl and CsCl) and an ammonium salt (CH3-(CH2)17-N-(CH3)3Br) are presented. The data indicate that the formation of a compact form of the double-stranded DNA in PEG-containing water-salt solutions depends not only on the PEG concentration and ionic strength but on tha cation nature as well. The compactization occurs most easily in the presence of Na+-ions. This indicates a specific character of interaction between Na+-ions and DNA phosphate groups which may be due to an optimum structural fit between the hydrated Na+-ions and orientation of the phosphate groups in the DNA molecule. The nature of forces involved in the processes of the intramolecular compactization and intermolecular aggregation of double-stranded DNA molecules in water-salt solution is discussed. The difference between the effect of Na+ and that of K+-ions on the compactization process at the ionic strengths close to physiological values makes it possible to suggest that the changes of the tertiary structure of double-stranded DNA which accompany its function in vivo may take place under conditions of a decreased water activity at the expense of relatively slight changes in ion composition of the water surrounding DNA.     

Electrostatic control of the overall shape of calmodulin: numerical calculations

The paper reports the results of numerical calculations of the pKa’s of the ionizable groups and the electrostatic interactions between calmodulin lobes in three different states of calmodulin: calcium-free, peptide-free; calcium-loaded, peptide-free; and calcium-loaded, peptide-bound. NMR and X-ray studies revealed that in these states the overall structure of calmodulin adopts various conformations referred as: disordered semi-compact, extended and compact conformations, respectively. In addition, a new X-ray structure was recently reported (Structure, 2003, 11, 1303) showing that calcium-loaded, peptide-free calmodulin can also adopt a compact conformation in addition to the well known extended conformation. The calculated energy changes of calcium-loaded, peptide-free calmodulin along the pathway connecting these two conformations provide a possible explanation for this structural plasticity. The effect of pH and organic compounds in the solution phase on the preference of calmodulin to adopt compact or extended conformations may be thus rationalized. Analysis of the contribution of the ionization changes to the energy of association of calmodulin lobes suggested that the formation of the compact forms requires protonation of several acidic residues. However, two different protonation scenarios are revealed: a protonation due to internal lobe organization and thus independent of the lobes association, and a protonation induced by the lobes association resulting to a proton uptake. In addition, the role of the individual residues on the energy of association of calmodulin lobes is calculated in two compact conformations (peptide-free and peptide-bound) and is shown that a set of residues always plays a dominant role in inter-domain interactions.     

Different effects of polylysine and polyarginine on the transition to a condensed state of DNA in polyethyleneglycol/salt solution.

Circular dichroism spectroscopy has been used to investigate the influence of polylysine and polyarginine on the transition to a condensed state of DNA brought about by high concentrations of polyethyleneglycol and salt. From the dependence on DNA concentration of the CD signals, the anomalous CD of free DNA in polyethyleneglycol/salt solution was attributed to the intermolecular association of DNA molecules. The CD spectral changes in polyethyleneglycol/salt solution of the DNA - polylysine complex were indistinguishable from those of free DNA while the DNA-polyarginine complex suffered much smaller spectral changes as compared with free DNA, at low DNA concentrations where time-independent CD spectra were observed in polyethyleneglycol/salt solution for both the complexed and free DNA. The repression of the spectral change by the latter complex was more remarkable at higher ratios of polyarginine to DNA. The facts indicate that, whereas polylysine binding has little influence on the intermolecular structural transition of double-stranded DNA into a compact molecular configuration in polyethyleneglycol/salt solution, polyarginine binding has an effect of inhibiting the transition.     

Electric birefringence of DNA and chromatin. Influence of divalent cations.

The effects of divalent cations on the DNA and chromatin conformation have been investigated by electric birefringence and birefringence relaxation measurements at low and constant ionic strength (0.001). An important decrease of the intrinsic optical anisotropy of DNA has been found in the presence of Mn2+ and Cu2+, but not with Mg2+. A complex variation of the mean relaxation time with the ratio I/P of ion to DNA-phosphate molar concentration has been evidenced in the presence of Mn2+ and Cu2+, while the mean relaxation time monotonously decreased in the presence of Mg2+. These observations are interpreted in terms of a specific organization of DNA in a compact, rigid structure, in the presence of Mn2+ and Cu2+, and a non-specific coiling in the presence of Mg2+. Drastic conformational changes encountered by chromatin in the presence of Mg2+ and Mn2+ cations have also been evidenced through electric birefringence measurements. They are interpreted by the formation of a superhelical compact arrangement of nucleosome strings which yielded a reversal of the birefringence sign with respect to the negative anisotropy observed in the presence of Na+ ions. The removal of the histone H1 prevented the appearance of this quaternary structure. More extended fragments of the chromatin chain obtained by ECTHAM chromatography of sonicated chromatin could not afford such compact arrangements.     

Relationship between the molecular structure of aqueous solutions of polyethylene glycol and the compactness of double-stranded DNA molecules

The dependence of viscosity of the water solutions of poly(ethylene glycol) (PEG) on the molecular weight has been studied. It has been shown that there is a "transitional" region in PEG properties which accounts for the formation of fluctuation polymer network of the PEG molecules. It has been shown that the "transitional" region in properties of PEG which appears at a certain concentration of PEG (CtrPEG) is characteristic of the PEG preparations with molecular weights exceeding 600 and dependence of the value of CtrPEG on the molecular weight of PEG was obtained. Compactization of double-stranded DNA molecules in PEG-containing water-salt solutions has been studied and the dependence of the value of CcrPEG, . i.e. the concentration of PEG at which the compact particles of DNA appear in the solution, on the molecular weight of PEG was obtained. The correlation between these two dependences reflecting quite different physico-chemical processes shows that the double-stranded DNA molecules are constrained within the polymer network of the PEG molecules. The influence of ionic strength and ionic composition of the solution on the formation of a compact form was investigated. The transition of the DNA molecules from a linear to a compact state may occur only at a definite value of ionic strength of the solution. This transition may occur at the change of K+ for Na+ cations (at a constant value of CPEG). The extent of compactization of the DNA molecules in PEG-containing water-salt solutions is monitored by the molecular structure and by the ionic strength of the solvent. It is supposed that the peculiarities of compactization of the DNA molecules in PEG-containing water-salt solutions reflect some characteristics of conformational transitions of the DNA molecules which occur in vivo.     

Effect of H+ on the K+ activation of adenosine-5'-monophosphate aminohydrolase.

The activation of adenosine-5'-monophosphate aminohydrolase from rabbit skeletal muscle by H+ has been demonstrated. Evidence is presented which indicates that the binding of H+ and K+ is linked, in that the dissociation constant (KA) for K+ activation is reduced as the pH is lowered. Concomitantly, the pK of several enzyme functional groups is changed when K+ is added to a solution of enzyme. This change is pK results in an uptake or release of H+, depending on the pH, and shows that K+ interacts with the enzyme to achieve its effect. The uptake or release of H+ provides a simple method of following conformational changes in the enzyme following interaction of K+. The KD for K+ interaction monitored by following pH changes is the same within experimental error as that measured from kinetic data.     

Cleavage of DNA adsorbed on the surface of phospholipid membranes by restrictases type II

Cleavage of phage lambda DNA by restriction endonucleases in the presence of model phosphatidylcholine membranes was studied. Bsp1, Pst1 and Bam H1 were found to cleave DNA under these conditions to a considerably decreased extent. This effect does not result from irreversible inactivation of the enzymes or their direct interaction with the membranes. The most probable explanation of the membrane inhibitory effect is the change of DNA substrate properties resulting from its Mg2+-mediated binding to the membranes.     

Raman microspectroscopic study of effects of Na(I) and Mg(II) ions on low pH induced DNA structural changes

In this work a confocal Raman microspectrometer is used to investigate the influence of Na(+) and Mg(2+) ions on the DNA structural changes induced by low pH. Measurements are carried out on calf thymus DNA at neutral pH (7) and pH 3 in the presence of low and high concentrations of Na(+) and Mg(2+) ions, respectively. It is found that low concentrations of Na(+) ions do not protect DNA against binding of H(+). High concentrations of monovalent ions can prevent protonation of the DNA double helix. Our Raman spectra show that low concentrations of Mg(2+) ions partly protect DNA against protonation of cytosine (line at 1262 cm(-1)) but do not protect adenine and guanine N(7) against binding of H(+) (characteristic lines at 1304 and 1488 cm(-1), respectively). High concentrations of Mg(2+) can prevent protonation of cytosine and protonation of adenine (disruption of AT pairs). By analyzing the line at 1488 cm(-1), which obtains most of its intensity from a guanine vibration, high magnesium salt protect the N(7) of guanine against protonation. A high salt concentration can prevent protonation of guanine, cytosine, and adenine in DNA. Higher salt concentrations cause less DNA protonation than lower salt concentrations. Magnesium ions are found to be more effective in protecting DNA against binding of H(+) as compared with calcium ions presented in a previous study. Divalent metal cations (Mg(2+), Ca(2+)) are more effective in protecting DNA against protonation than monovalent ions (Na(+)).     

Attachment of Pseudomonas fluorescens to glass and influence of electrolytes on bacterium-substratum separation distance.

The influence of Na+, Ca2+, La3+, and Fe3+ on the adhesion of Pseudomonas fluorescens H2 and H2S was investigated with interference reflection microscopy (IRM). IRM is a light microscopy technique which allows (i) visualization of the adhesive sites of living bacteria as they attach to a glass cover slip surface and (ii) evaluation of the bacterium-glass surface separation distance within a range of 0 to ca. 100 nm. The addition of each cation caused changes in IRM images consistent with a decrease in the separation distance, and minimum effective concentrations were as follows: Na+, 1 mM; Ca2+, 1 mM; La3+, 50 microM; and Fe3+, 50 microM. With strain H2, the effects of Na+, Ca2+, and La3+ were fully reversible in that the separation distance increased again when the electrolyte was replaced with distilled water. However, with strain H2S, a spontaneous mutant of H2 with increased attachment ability, only the effect of Na+ was fully reversible, and the effects of Ca2+ and La3+ were only partially reversible or irreversible. The effect of Fe3+ was irreversible with both strains, but this may be related not only to the electrolytic nature of Fe3+ but also to the decrease in solution pH to 3.5 caused by its addition. It is proposed that the electrolytes caused a decrease in separation distance by neutralizing negative charges on bacterial surface polymers and that the different effects obtained with the two strains are related to their different adhesion abilities.     

Study of chromatin organization with trypsin immobilized on collagen membranes

Trypsin immobilized on collagen membranes has been used to digest chromatin polynucleosomes. With this method, the use of protease inhibitor is avoided and the digestion time easily controlled simply by taking the membrane out of the chromatin solution. Its most fundamental advantage is however to allow the mild removing of the most accessible histone fragments without addition of salt then without perturbation of their ionic environment. Degradation of histone fractions were correlated with conformational changes using circular dichroism and electric birefringence measurements. On digestion, the sign of birefringence reversed, becoming negative, and an increase of molar ellipticity was observed. These changes reflecting the unfolding of DNA correspond to the digestion of H1 and also of fragments of H3. This would indicate that H3 and particularly its basic terminal regions, play a fundamental role in the maintenance of chromatin in a compact structure.     

An octamer of histones H3 and H4 forms a compact complex with DNA of nucleosome size.

Equimolar mixtures of histones H3 and H4 have been reconstituted onto DNA of nucleosome core size. Two distinct complexes are formed in a relative abundance that depends on the starting ratio of H3 + H4 to DNA. One of these complexes contains two H3-H4 dimers for each DNA molecule, and has a sedimentation coefficient of 7.5S. The other complex contains an octamer consisting of four H3-H4 dimers, and has a sedimentation coefficient of 10.4S. On the basis of these measurements, we conclude that the octamer complex (but not the tetramer complex) is a fully folded, compact structure resembling the nucleosome.     

Kinetics and equilibria for the formation of a new DNA metal-intercalator: the cyclic polyamine Neotrien/copper(II) complex

A study has been performed of the kinetics and equilibria involved in complex formation between the macrocyclic polyamine 2,5,8,11-tetraaza[12]-[12](2,9)[1,10]-phenanthrolinophane (Neotrien) and Cu(II) in acidic aqueous solution and ionic strength 0.5 M (NaCl), by means of the stopped-flow method and UV spectrophotometry. Spectrophotometric titrations and kinetic experiments revealed that the binding of Cu(II) to Neotrien gives rise to several 1:1 complexes differing in their degree of protonation. Under the experimental hydrogen ion concentration range investigated, complexation occurs by two parallel paths: (a) M2+ + (H4L)4+ <==> (MH4L)6+ and (b) M2+ + (H3L)3+ <==> (MH3L)5+. The rate constants values found for complex formation, by paths (a) and (b), are much lower than the values expected from water exchange at copper(II) and other amine/Cu(II) complexation kinetic constants. Kinetic experiments at different NaCl concentrations indicated that this finding was not due to chloride ion competition in complex formation with Neotrien, but it was related to a ring rigidity effect. As the phenanthroline moiety could, in principle, interact with nucleic acids by intercalation or external binding, some preliminary measurements concerned with the possible interactions occurring between the Cu(II)/Neotrien complex and calf thymus DNA (CT-DNA) have also been carried out. The absorption spectra of the Cu(II)/Neotrien complex change upon addition of CT-DNA at pH 7.0, revealing the occurrence of complex-nucleic acid interactions. Moreover, fluorescence titrations, carried out by adding the Cu(II)/Neotrien complex to CT-DNA, previously saturated with ethidium bromide (EB), show that the Cu(II)/Neotrien complex is able to displace EB from DNA, suggesting the complex is able to intercalate into the polynucleotide and then to cleave the phosphodiester bond of DNA.     

Studies on conformational changes in the DNA structure induced by protonation: reversible and irreversible acid titrations and sedimentation measurments

Reversible and irreversible conformational changes in the acid-induced denaturation of DNA were studied by spectrophotometric titration, sedimentation, and melting measurements. A GC-rich DNA (72 mole-%) shows complete or partial reversibility of the titration profiles within the pH region of transition from helix to coil, while AT-rich DNA (29 mole-%) is irreversible in its titration behavior at each acid pH below the onset of the transition. The results for GC-rich DNA further indicate distinct differences in the titration behavior, which can be attributed to differences in the frequency of GC clusters along the DNA molecule. Plots of the sedimentation coefficient and the parameter a_s^app against pH lead to the conclusion that conformational changes occur before the onset of the acid-induced helix–coil transition. These alterations are more pronounced upon protonation of larger GC-rich domains than of smaller ones, as concluded from very marked differences observed in the sedimentation–pH behavior of two GC-rich DNA's. An acid denaturation scheme for a GC-rich DNA segment is suggested. Reversibility of the acid denaturation is explained by the existence of stable, protonated, single GC base pairs in nonprotonated stacked single-stranded domains formed in the acid-induced transition region.     

Effect of ethanol on structural transitions of DNA and polyphosphates under Ca2+ ions action in mixed solutions

In the present work using the IR spectroscopy method the effect of ethanol on structural transitions of DNA and polyphosphates under the action of Ca2+ ions in mixed solutions containing ethanol (0-25 vol.%) was studied. It was shown that, on its interaction with Ca2+ ions, in aqueous and mixed solutions DNA becomes transformed into compact form. With the increase of concentration of ethanol the degree of Ca2+-induced DNA compactisation rises. It was found that, in mixed solutions containing ethanol, Ca2+-induced DNA compactisation depends not only on the solution's dielectric permeability but also on the solution structure. On stabilisation of the water structure in the presence of low ethanol concentrations a stabilisation of the DNA macromolecule occurs that leads to the increase of the Ca2+ ion concentration necessary for DNA compactisation. Comparison of the effects of ethanol on Ca2+-induced structural transitions in DNA and polyphosphates in mixed solvents permits to suppose that at alcohol concentrations in solution resulting in disruption of the water spatial structure, some peculiarities are observed in the behavior of those molecules whose hydrophobic interactions are essential.     

A comparative study of the interaction of 5,10,15,20-tetrakis (N-methylpyridinium-4-yl)porphyrin and its zinc complex with DNA using fluorescence spectroscopy and topoisomerisation.

Binding of 5,10,15,20-tetrakis (N-methylpyridinium-4-yl)porphyrin (H2TMPyP4+) and its zinc complex (ZnTMPyP4+) to DNA is demonstrated by their coelectrophoresis and by absorption and fluorescence spectroscopic methods. Topoisomerisation of pBR322 DNA shows that H2TMPyP4+ unwinds DNA as efficiently as ethidium bromide showing that it intercalates at many sites. ZnTMPyP4+ may cause limited unwinding. Marked changes in the fluorescence spectra of the porphyrins are found in the presence of DNA. The fluorescence intensity of either H2TMPyP4+ or ZnTMPyP4+ is enhanced in the presence of poly (d(A-T)), whereas in the presence of poly (d(G-C] the fluorescence intensity of ZnTMPyP4+ is only slightly affected and that of H2TMPyP4+ markedly reduced. Both the porphyrins photosensitize the cleavage of DNA in aerated solution upon visible light irradiation.