Thermal stability of the Z-conformation of the tetranucleoside triphosphate (m5dC-dG)2

The tetranucleoside triphosphate d(m5C-G)2 has been studied in solution by circular dichroism and 31P nuclear magnetic resonance as a function of temperature, in presence of 3 M NaClO4. It is shown that in such high ionic strength d(m5C-G)2 may adopt a Z-like conformation for temperatures lower than 5 degrees C. At these temperatures, another conformation, in slow equilibrium with the Z-like one, is also detected. Increasing the temperature leads to a transition from the Z-like conformation to intermediate forms before melting. It is demonstrated that these intermediates are not the B form.     

Spectroscopic studies of (m5dC-dG)3: thermal stability of B- and Z-forms.

The hexanucleoside pentaphosphate d(m5CpGpm5CpGpm5CpG) has been studied in solution by ultra-violet absorption, circular dichroism and 31P nuclear magnetic resonance under various experimental conditions. In 0.2 M NaClO4 at low temperature, an hexamer duplex is formed which has a B or B-like conformation. As the salt concentration is increased, a transition from a B-form to the Z-form occurs and is complete in 3 M NaClO4. In 3 M NaClO4, the behavior of the Z double helix is complex as a function of temperature. The variation of the circular dichroism at 295 nm is biphasic. A first transition occurs over a large range of temperature and corresponds to a conformational change due to a non-cooperative intramolecular process. Ultra-violet absorption and 31P nuclear magnetic resonance show that the new conformation arising from a distortion of the backbone is not similar to that observed in low salt conditions (B-form). At high hexanucleotide concentration, aggregates are formed. The second transition is cooperative and corresponds to the melting of a double stranded helix into single strands.     

A study of the thermal stability of the Z form of (m5dC-dG)3 by resonance Raman spectroscopy.

The thermal stability of the hexanucleoside pentaphosphate m5dCpdGpm5dCpdGpm5 dCpdG has been studied by resonance Raman spectroscopy with 257 nm excitation wavelength. At low temperature and in 3M NaClO4, the Raman spectrum resembles that of poly(dG-dC).poly(dG-dC) in the Z conformation. As the temperature is increased, the position and the intensity of several bands (1312 cm-1, 1482 cm-1, 1584 cm-1 and 1632 cm-1) are modified. The variation of intensity versus temperature is biphasic. Analysis of the results suggests that the increase of temperature induces first a transition from the Z form to an intermediate stable form which then melts. These results and those previously obtained by circular dichroism and 31P nuclear magnetic resonance suggest that the intermediate form belongs to the left family but with changes in the stacking of the bases and the geometry of the phosphate groups as compared to the canonical Z form.     

Thermodynamics of B-Z transition of poly[d(G-C)] in a water-ethanol solution. The "tie calorimetry"

Poly[d(G-C)] in a 55% ethanol solution undergoes a transition from the Z form to the B form when the temperature is increased from 20 degrees to 50 degrees C. The enthalpy of the transition, delta HBA = -1.4 kcal/mol, has been determined with a "tie" polyamine which stabilizes the Z conformation. This value has been shown to be practically independent of ionic strength within the range of 5 X 10(-4) M - 2 X 10(-3) M NaCl.     

Non-contiguous regions of Z-DNA in a DNA dodecamer.

The conformation of the self-complimentary DNA dodecamer d(br5CGbr5CGAATTbr5CGbr5CG) has been investigated in a variety of salt and solvent conditions by one and two-dimensional 1H NMR. In low salt aqueous solutions, the molecule forms a regular B-DNA structure similar to the unmodified dodecamer. However, in aqueous solution containing high salt concentration and methanol, the dodecamer adopts a structure in which the br5CGbr5CG ends of the molecule are in a Z-DNA like conformation and the AATT region is neither standard B-DNA nor Z-DNA. The implications of these results for the structure of junctions between B and Z-DNA and the sequence specificity of Z-DNA are discussed.     

1H NMR and circular dichroism studies of the B and Z conformations of the self-complementary deoxyhexanucleotide d(m5C-G-C-G-m5-C-G): mechanism of the Z-B-coil transitions

The double-helical conformations of d(m5-C-G-C-G-m5-C-G) in aqueous solution were studied by circular dichroism and 1H NMR spectroscopy. In 0.1 M NaCl, only the B form is detected whereas the Z form is strongly predominant in 3 M NaCl. In the presence of 2 M NaCl, two resonance signals corresponding to the B and Z duplexes were observed for each proton below 50 degrees C, indicating a slow exchange between B and Z. However, the B-Z exchange becomes intermediate or fast in the 55-80 degrees C temperature interval. By contrast the exchange between B helix and single-stranded (or coil) forms is much faster for the same temperature conditions. The Z form is only detectable when the coil form is practically absent. With decreasing temperature the B form decreases in favor of the Z form. From proton line-width measurements under various experimental conditions, it was also shown that Z exchanges only with B, while the latter also exchanges with the single-stranded form (S): Z in equilibrium B in equilibrium S. The enthalpy value is about 8 +/- 1 kcal/mol for the B-Z transition and about 40 +/- 2 kcal/mol for the B-S dissociation (2 M NaCl solution). The activation energy is about 47 +/- 2 kcal/mol for the Z----B and 39 +/- 2 kcal/mol for the B----Z reaction. Very good agreement between the experimental results and computed data (based on the above kinetic reaction model) was found for the B, Z, and coil proportions. The B-Z transition of methylated d(C-G)n oligomers is only possible when the Watson-Crick hydrogen bonds between the CG base pairs are firmly maintained; otherwise, the transformation from B to Z would not occur, and B-S dissociation would take place instead.     

Heat denaturation of immunoglobin G in monomolecular layers

Studies were carried out of viscous-elastic properties of monomolecular layers of human immunoglobulin IgG formed at the interface water solutions of NaCl--air within 20 degrees C to 50 degrees C at NaCl concentration in sublayer from 0.3 to 1.0 M. It has been shown that at concentrations from 0.3 to 0.5 M of NaCl IgG macromolecules keep the tertiary structure, and in the region 35 +/- 5 degrees C a conformation transition is observed. The recorded conformation transition is reversible and of entropy nature. At NaCl concentration 0.75 in the sublayer and temperature close to 35 degrees C IgG macromolecules undergo irreversible structural changes due to the destruction of hydrogen and disulfide bonds in IgG molecules. Macromolecules dissociate to fragments with molecular mass 49,000 +/- 2000. At NaCl concentration 1.0 M and temperatures 30-50 degrees C IgG macromolecules of the monolayer are in a dissociated state. Changes in entropy, enthalpy and heat capacity as well as areas occupied by the macromolecules at dense packing, molecular mass and efficient electric dipole moment of the monolayer are calculated.     

Partially folded states of the cytolytic protein sticholysin II

Sticholysin II (Stn II) is a cytolytic protein produced by the sea anemone Stichodactyla helianthus, its effect being related to pore formation. The conformation of the protein and its temperature-induced transitions, in the 1.5-12.0 pH range and in the 0-0.5 M NaCl concentration interval, have been studied by circular dichroism and fluorescence spectroscopy. At temperature < 35 degrees C, the protein maintains the same, high beta-structure content, folded conformation in the 1.5-11.0 pH range and ionic strength up to 0.5 M. In the 1.5-3.5 pH range and ionic strength > or = 0.1 M, Stn II shows a thermal transition, resulting in a partially folded state characterized by: (i) a native-like content of regular secondary structure, as detected by far-UV CD; (ii) a largely disordered tertiary structure, as detected by near-UV CD, with partially exposed tryptophan residues according to their fluorescence emission; and (iii) ability to bind the hydrophobic probe 2-anilinonaphthalene-6-sulfonic acid. In the pH range 4.0-10.5, thermally-induced protein aggregation occurs. The obtained results demonstrate the existence of partially folded state of Stn II, which may contribute to the pore formation ability of this cytolysin.     

L-nucleotides and 8-methylguanine of d(C1m8G2C3G4C5LG6LC7G8C9G10)2 act cooperatively to promote a left-handed helix under physiological salt conditions

The structure and thermal stability of a hetero chiral decaoligodeoxyribonucleotide duplex d(C1m8 G2C3G4C5LG6LC7G8C9G10)d(C11m8G12C13G14C15LG16LC17G18C19G20) (O1) with two contiguous pairs of enantiomeric 2'-deoxy-L-ribonucleotides (C5LG6L/C15LG16L) at its centre and an 8-methylguanine at position 2/12 was analysed by circular dichroism, NMR and molecular modelling. O1 resolves in a left-handed helical structure already at low salt concentration (0.1 M NaCl). The central L2-sugar portion assumes a B* left-handed conformation (mirror-image of right-handed B-DNA) while its flanking D4-sugar portions adopt the known Z left-handed conformation. The resulting Z4-B2*-Z4 structure (left-handed helix) is the reverse of that of B4-Z2*-B4 (right-handed helix) displayed by the nearly related decaoligodeoxyribonucleotide d(mC1G2mC3G4C5L G6LmC7G8mC9G10)2, at the same low salt concentration (0.1 M NaCl). In the same experimental conditions, d(C1m8G2C3G4C5G6C7G8C9G10)2 (O2), the stereoregular version of O1, resolves into a right-handed B-DNA helix. Thus, both the 8-methylguanine and the enantiomeric step CLpGL at the centre of the molecule are needed to induce left-handed helicity. Remarkably, in the various heterochiral decaoligodeoxyribonucleotides so far analysed by us, when the central CLpGL adopts the B* (respectively Z*) conformation, then the adjacent steps automatically resolves in the Z (respectively B) conformation. This allows a good optimisation of the base-base stackings and base-sugar van der Waals interactions at the ZB*/B*Z (respectively BZ*/Z*B) junctions so that the Z4-B2*-Z4 (respectively B4-Z2*-B4) helix displays a Tm (approximately 65 degrees C) that is only 5 degrees C lower than the one of its homochiral counterpart. Here we anticipate that a large variety of DNA helices can be generated at low salt concentration by manipulating internal factors such as sugar configuration, duplex length, nucleotide composition and base methylation. These helices can constitute powerful tools for structural and biological investigations, especially as they can be used in physiological conditions.     

Toroidal condensation of Z DNA and identification of an intermediate in the B to Z transition of poly(dG-m5dC) X poly(dG-m5dC)

Using a combination of spectroscopic techniques, quasi-elastic laser light scattering (QLS), and electron microscopy (EM), we have been able to show that the B to Z transition of poly(dG-m5dC) X poly(dG-m5dC) is accompanied by extensive condensation of the DNA in both low and high ionic strength buffers. At low concentrations of NaCl (2 mM Na+), an intermediate rodlike form, which exhibits a circular dichroism (CD) spectrum characteristic of an equimolar mixture of B and Z forms, is observed. This is produced by the orderly self-association of about four molecules of the polymer after prolonged incubation of a concentrated solution at 4 degrees C. On addition of 5 microM Co(NH3)63+, the CD spectrum of the intermediate changes to that of the Z form, which is visualized as a dense population of discrete toroids on an EM grid stained with uranyl acetate. On the other hand, addition of NaCl to a solution of poly(dG-m5dC) X poly(dG-m5dC) in the absence of any multivalent ion condenses the polymer to toroidal structures at the midpoint (0.75 M NaCl) of the B to Z transition. Further addition of NaCl unfolds these toroids to rodlike structures, which show characteristic Z-form CD spectra. These results show that Z DNA can take up a variety of tertiary structural forms and indicate that its inverted CD spectrum is due to its left-handed helical sense rather than to differential scattering artifacts.     

Two-dimensional 1H NMR study of d(br5C-G)3 in the Z-form. Self association and flexibility of the left-handed double helix

The Z conformation of the auto complementary hexanucleoside pentaphosphate d(br5C-G)3 in 1 M NaClO4 solution has been investigated by using 2D NMR techniques. NOESY experiments performed at different temperatures show that the oligonucleotide exhibits end-to-end associations at room temperature. The conformation of the hexanucleotide molecules is very similar to that found in the crystal which was described by Chevrier et al. (J. Mol. Biol., 1986, 188, 707-719) as a Z-I form. When the temperature is increased the aggregates are dissociated and a conformational change is observed which is interpreted as a Z-I in equilibrium Z-II transition.     

Stabilization of Z-RNA by chemical bromination and its recognition by anti-Z-DNA antibodies

Limited chemical bromination of poly[r(C-G)] (32% br8G, 26% br5C) results in partial modification of guanine C8 and cytosine C5, producing a mixture of A- and Z-RNA forms. The Z conformation in the brominated polynucleotide is stabilized at much lower ionic strength than in the unmodified polynucleotide. More extensive bromination of poly[r(C-G)] (greater than 49% br8G, 43% br5C) results in stabilization of a form of RNA having a Z-DNA-like (ZD) CD spectrum in low-salt, pH 7.0-7.5 buffers. Raising the ionic strength to 6 M NaBr or NaClO4 results in a transition in Br-poly[r(C-G)] to a Z-RNA (ZR) conformation as judged by CD spectroscopy. At lower ionic strength Z-DNA-like (ZD) and A-RNA conformations are also present. 1H NMR data demonstrate a 1/1 mixture of A- and Z-RNAs in 110 mM NaBr buffer at 37 degrees C. Nuclear Overhauser effect (NOE) experiments permit complete assignments of GH8, CH6, CH5, GH1', and CH1' resonances in both the A- and Z-forms. GH8----GH1' NOEs demonstrate the presence of both A- and Z-form GH8 resonances in slow exchange on the NMR time scale. The NMR results indicate that unbrominated guanine residues undergo transition to the syn conformation (Z-form). Raman scattering data are consistent with a mixture of A- and Z-RNAs in 110 mM NaCl buffer at 37 degrees C. Comparison with the spectrum of Z-DNA indicates that there may be different glycosidic torsion angles in Z-RNA and Z-DNA [Tinoco, I., Jr., Cruz, P., Davis, P., Hall, K., Hardin, C. C., Mathies, R. A., Puglisi, J. D., Trulson, M. O., Johnson, W. C., & Neilson, T. (1986) in Structure and Dynamics of RNA, pp 55-68, Plenum, New York].(ABSTRACT TRUNCATED AT 250 WORDS)     

New DNA polymorphism: evidence for a low salt, left-handed form of poly(dG-m5dC).

Spectroscopic studies on solutions of poly(dG-m5dC) over a wide range of salt concentration are presented. Low salt solutions [( Na+]) less than 2 mM) of poly(dG-m5dC) produce circular dichroism (CD) spectra typical of the left-handed, Z form at high salt [( Na+] = 1.75 M). Solutions of poly(dG-m5dC) at intermediate salt concentrations, e.g., 142 mM, yield CD spectra characteristic of the right-handed, B conformation. 31p NMR spectra of the low salt form of poly(dG-m5dC) reveal two well separated peaks, split by 1.4 ppm, consistent with a dinucleotide repeat. Kinetic studies show that the transition from the low salt form to teh right-handed B form is slow, as expected for a major conformational change. These results suggest that the Z conformation in poly(dG-m5dC) can be stabilized at very low salt as well as at high salt.     

Conformational lability of poly(dG-m5dC):poly(dG-m5dC).

The remarkable conformational lability of poly(dG-m5dC):poly(dG-m5dC) is demonstrated by the observation of an acid-mediated conformational hysteresis. An acid-mediated Z conformation that exists in solutions containing low sodium concentrations that would normally favor the B conformation is described in this report. This Z conformation is reached by an acid-base titration of a B-poly(dG-m5dC):poly(dG-m5dC) solution which is not far from the B-Z transition midpoint. The resulting Z conformation is thermally very stable, with direct melting into single strands at approximately 100 degrees C. In contrast, the B form DNA, initially in solutions of the same ionic strength but without exposure to acidic pH, exhibits a biphasic melting profile, with conversion into the Z form (with high cooperativity) prior to an eventual denaturation into single strands at around 100 degrees C. Cooling experiments reveal that such biphasic transitions are quite reversible. The transition midpoint for the thermally poised B to Z transformation depends strongly on the NaCl concentration and varies with sample batch. The acid-mediated Z form binds ethidium more weakly than its B counterpart, and the ethidium induced Z to B conversion occurs in a step-wise (non-allosteric) fashion without the requirement of a threshold concentration. The acid-mediated as well as the thermally poised Z conformations are reversed by the addition of EDTA, suggesting the involvement of trace amounts of multivalent metal ions.     

Temperature and pH dependent changes of immunoglobulin G structure.

1. The temperature and pH functions of the myeloma IgG(K) conformation were studied by optical rotatory dispersion, circular dichroism, thermal perturbation difference spectroscopy, solvent perturbation difference spectroscopy, electrochemical iodination and difference adiabatic scanning microcalorimetry. 2. The IgG studied was found to be capable of a fully reversible structural change between pH 6.5 and 6.0. A transition occurring at low pH is accompanied by an increase of exposure of the chromophores to the solvent. 3. The "alkaline state" was found to be capable of a fully reversible S-like transition at temperatures between 25 and 35 degrees C. The changes occurring at the higher temperature are accompanied by the screening of 14-15 tyrosine residues and probably by a small increase in the helicity of the protein. These changes are not accompanied by an appreciable heat effect. The thermal denaturation of the "alkaline state" occurs only at 64 degrees C in the narrow temperature interval (3-4 degrees C). 4. The "acid state" is not accompanied by S-like transition at 25-35 degrees C. The thermal denaturation of the "acid state" occurs at 54 degrees C in the wide temperature interval (8-9 degrees C). 5. It was proposed that the ionisation of the invariant histidine residues situated in the "cavity" between the constant and variable domains causes the pH transition studied. The temperature changes in the interval 25-35 degrees C are explained by the alteration of the domains interposition. Similar alterations were investigated as a result of antigen-antibody reaction.     

Mn2+ and other transition metals at low concentration induce the right-to-left helical transformation of poly[d(G-C)].

The effects of the first-row transition metal ions on the right(B)- to left(Z)-handed helical transition of poly[d(G-C)] have been determined. The Z conformation is induced by MnCl2 at submillimolar concentrations. The forward reaction has a very large activation energy (440 kJ/mol) so that a facile conversion occurs only at temperatures above 45 degrees C. However, the left-handed form remains stable upon cooling. The addition of ethanol (20% v/v) eliminates the requirement for elevated temperature. The transition is highly co-operative and is accompanied by spectral changes (absorption, circular dichroism) characteristic for the B----Z conformational transition. NiCl2 and CoCl2 also induce the B----Z transition in poly[d(G-C)] but the activation energies and thus the temperature requirements for the forward reaction are lower than those observed with MnCl2. The left-handed DNA formed in the presence of Mn2+ is similar to 'Z DNA' previously described in Mg2+-EtOH (van de Sande and Jovin , 1982): (a) it readily sediments out of solution at low speed as a consequence of intermolecular association which, however, is not accompanied by turbidity; and (b) it supports the binding of ethidium bromide although this drug interacts preferentially with the B form of DNA. With Ni2+, the B----Z isomerization step can be separated from the subsequent specific Z----Z* association. Mn2+, Ni2+, and Co2+ also promote the B----Z transition of poly[d(G-m5C)] at substoichiometric concentrations with respect to DNA nucleotide.     

The B goes to Z transition of poly(dG-dC) . poly(dG-dC) modified by some platinum derivatives.

Poly(dG-dC) . poly(dG-dC) was modified by chlorodiethylenetriamino platinum (II) chloride, cis-dichlorodiammine platinum (II) and trans-dichlorodiammine platinum (II), respectively. The conformation of these modified poly(dG-dC) . poly(dG-dC) was studied by circular dichroism. In 4 M Na+, the circular dichroism spectra of poly(dG-dC)dien-Pt (0 less than or equal to rb less than or equal to 0.2) are similar (rb is the amount of bound platinum per base). It is concluded that the conformation of these polymers belongs to the Z-family. Dien-Pt complexes stabilize the Z-form. The midpoint of the Z goes to B transition of poly(dG-dC)dien-Pt(0.12) is at 0.2 M NaCl. Moreover another B goes to Z transition is observed at lower salt concentration (midpoint at 6 mM NaCl). In 1 mM phosphate buffer, the stability of Z-poly(dG-dC)dien-Pt(0.12) is greatly affected by the presence of small amounts of EDTA. Poly(dG-dC) . poly(dG-dC) modified by cis-Pt and trans-Pt complexes do not adopt the Z-form even in high salt concentration.     

Physical characterization of a kinetoplast DNA fragment with unusual properties

A 414-base pair fragment from a Leishmania tarentolae kinetoplast DNA minicircle has unusual physical properties. We reported previously that in comparison to phi X174 and pBR322 control fragments, the kinetoplast fragment behaves in gel electrophoresis, gel filtration, and electric dichroism experiments as if it has an unusually compact conformation. We accounted for these unusual properties by proposing that the fragment is a systematically bent helix (Marini, J.C., Levene, S.D., Crothers, D.M., and Englund, P.T. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 7664-7668). In this paper, we further explore the properties of the kinetoplast fragment. Because of its compact conformation, the kinetoplast fragment has difficulty in snaking through polyacrylamide gels and therefore migrates unusually slowly in electrophoresis experiments. Warming (53 degrees C) and ethanol (5-20%) partially normalize gel migration; glyoxal treatment results in denatured strands with electrophoretic mobility close to that expected for their size. In vivo modification does not appear to be responsible for the fragment's properties; its anomalous electrophoretic behavior persists after proteinase K treatment, phenol extraction, or after cloning into pBR322 and reisolation. Velocity sedimentation experiments rule out fragment aggregation. Secondary structure, such as a cruciform, is not detectable by S1 or mung bean nuclease digestion. The kinetoplast fragment has circular dichroism spectra characteristic of a B-type helix. With increasing temperature, there is an increase in the 270/280 ellipticity ratio. Circular dichroism spectra taken in the presence of ethanol show a B to A helix transition at unusually low ethanol concentrations (between 44 and 54% (w/w]. Thermal denaturation reveals a triphasic melting curve.     

Temperature-dependent beta-sheet formation in beta-amyloid Abeta(1-40) peptide in water: uncoupling beta-structure folding from aggregation

To probe the role of temperature in the conversion of soluble Alzheimer's beta-amyloid peptide (Abeta) to insoluble beta-sheet rich aggregates, we analyzed the solution conformation of Abeta(1-40) from 0 to 98 degrees C by far-UV circular dichroism (CD) and native gel electrophoresis. The CD spectra of 15-300 microg/ml Abeta(1-40) in aqueous solution (pH approximately 4.6) at 0 degrees C are concentration-independent and suggest a substantially unfolded and/or unusually folded conformation characteristic of Abeta monomer or dimer. Heating from 0 to 37 degrees C induces a rapid reversible coil to beta-strand transition that is independent of the peptide concentration and thus is not linked to oligomerization. Consequently, this transition may occur within the Abeta(1-40) monomer or dimer. Incubation at 37 degrees C leads to slow reversible concentration-dependent beta-sheet accumulation; heating to 85 degrees C induces further beta-sheet folding and oligomerization. Our results demonstrate the importance of temperature and thermal history for the conformation of Abeta.