Thermal stability of the Z conformation of the hexanucleoside pentaphosphate d(br5CGbr5CGbr5CG): evidence for a conformational transition before melting

The thermal stability of the hexanucleoside pentaphosphate d(br5CGbr5CGbr5CG) has been studied at two nucleotide concentrations, in the presence of 1 M NaClO4. At low nucleotide concentration (7 X 10(-5) M), circular dichroism experiments show a conformational transition from the Z conformation to another conformation, named X, which is not the B conformation, as the temperature is increased from 0 to 35 degrees C. Between 40 and 65 degrees C, another transition is observed which corresponds to the melting of the X conformation. At higher nucleotide concentration (2 X 10(-3) M), circular dichroism and 31P nuclear magnetic resonance experiments show that at low temperature (br5dC-dG)3 adopts the Z conformation. There are associations between the oligonucleotides which progressively disappear as the temperature increases. In the range 35-60 degrees C a transition from the Z conformation to another conformation is observed. This new conformation is the X conformation detected at low nucleotide concentration.     

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.     

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.     

The tetraribonucleotide rCpGpCpG forms a left-handed Z-RNA double-helix.

NMR and circular dichroism studies show that the RNA tetranucleotide rCpGpCpG can form a Z-RNA left-handed double-helix. In 1.0 M NaClO4, circular dichroism measurements indicate that the tetramer is in the A-form. In 6.0 M NaClO4, there is a characteristic change in the circular dichroism, indicating that the tetramer adopts a left-handed Z-form. This conformation is verified by phosphorus and proton NMR studies. The 31P spectrum shows a large downfield shift in one of the resonances upon an increase in salt concentration. Proton nuclear Overhauser effect (NOE) experiments indicate that the guanosines are in the syn conformation. These results are consistent with the formation of a Z-form double-helix.     

Comparison of the B- and Z-form hairpin loop structures formed by d(CG)5T4(CG)5

The partially self-complementary synthetic DNA oligonucleotide d(CG)5T4(CG)5 has been studied by using 1H and 31P NMR and circular dichroism. Results show that, under low-salt conditions (120 mM NaCl buffer), an intramolecular hairpin loop exists in which the double-helical stem region is B-form and the thymidine loop residues have predominantly southern (C2'-endo) sugar conformations. The thymidine glycosidic torsion angles are intermediate between syn and anti or exist as an equilibrium mixture of residues in the two extremes. NOESY data indicate that the structure of the loop region is very similar to that found for d(CG)2T4(CG)2 [Hare, D. R., & Reid, B. R. (1986) Biochemistry 25, 5341-5350]. Under high-salt conditions (6 M NaClO4 buffer), the dominant form (approximately equal to 85%) is an intramolecular hairpin structure in which the stem region forms a Z-form double helix. As in the B-form, the loop thymidine residues are intermediate between the syn and anti conformations or exist as an equilibrium mixture of the two, but the thymidine sugar conformations differ in that they are biased toward northern (C3'-endo) conformations.     

Acid titrations of poly(dG-dC).poly(dG-dC) in aqueous solution and in a w/o microemulsion

The model polynucleotide poly(dG-dC).poly(dG-dC) (polyGC) was titrated with a strong acid (HCl) in aqueous unbuffered solutions and in the quaternary w/o microemulsion CTAB/n-pentanol/n-hexane/water. The titrations, performed at several concentrations of NaCl in the range 0.005 to 0.600 M, were followed by recording the modifications of the electronic absorption and of the CD spectra (210< or = lambda < or =350 nm) upon addition of the acid. In solution, the polynucleotide undergoes two acid-induced transitions, neither of which corresponds to denaturation of the duplex to single coil. The first transition leads to the Hoogsteen type synG.C+ duplex, while the second leads to the C+.C duplex. The initial B-form of polyGC was recovered by back-titration with NaOH. The apparent pKa values were obtained for both steps of the titration, at all salt concentrations. A reasonably linear dependence of pKa1 and pKa2 from p[NaCl] was obtained, with both pKa values decreasing with increasing ionic strength. In microemulsion, at salt concentrations < or = 0.300 M, an acid-induced transition was observed, matching the first conformational transition recorded also in solution. However, further addition of acid led to denaturation of the protonated duplex. Renaturation of polyGC was obtained by back-titration with NaOH. At salt concentrations > 0.300 M, polyGC is present as a mixture of B-form and psi- aggregates, that slowly separate from the microemulsion. The acid titration induces at first a conformational transition similar to the one observed at low salt or in solution, then denaturation occurs, which is however preceded by the appearance of a transient conformation, that has been tentatively classified as a left-handed Z double helix.     

Immunological and spectroscopic studies of poly(dG-dC).poly(dG-dC) modified by cis-diamminedichloroplatinum(II)

The conformational changes induced by the binding of cis-diamminedichloroplatinum(II) to poly(dG-dC).poly(dG-dC) have been studied by reaction with specific antibodies, by circular dichroism and 31P nuclear magnetic resonance. Polyclonal and monoclonal antibodies to Z-DNA bind to platinated poly(dG-dC).poly(dG-dC) at low and high ionic strength. Antibodies elicited in rabbits immunized with the platinated polynucleotide bind to double stranded polynucleotides known to adopt the Z-conformation. At low and high ionic strength the circular dichroism spectrum of platinated poly(dG-dC).poly(dG- dC) does not resemble that of poly(dG-dC).poly(dG-dC) (B or Z conformation). At low ionic strength, the characteristic 31P nuclear magnetic resonance spectrum of the Z-form is not detected. It appears only at high ionic strength, as a component of a more complex spectrum.     

Formation of a left-handed RNA double helix: energetics of the A-Z transition of poly[r(G-C)] in concentrated NaClO4 solutions

Ultraviolet spectroscopic and nuclear magnetic resonance (NMR) studies have shown that poly[r(G-C)] in a solution of 4 M NaClO4 undergoes a transition to a left-handed Z-RNA helix upon raising the temperature to 60 degrees C [Hall, K., Cruz, P., Tinoco, I., Jr., Jovin, T. M., & van de Sande, J. H. (1984) Nature (London) 311, 584-586]. In the present report, the transition temperature of this particular order/order transition is shown to increase with decreasing NaClO4 concentration to about 110 degrees C, above which only the helix-to-random coil transition is detectable. The reversibility and cooperativity of the helix/helix conversion has facilitated the quantitative evaluation of the transition enthalpy by means of differential scanning microcalorimetry. In 5 M NaClO4, the transition temperature is 43 degrees C, the conversion enthalpy 4.2 kJ (1.0 kcal) per mole of base pair, and the corresponding entropy change 13 J (3.1 cal) deg-1. The van't Hoff enthalpy for the same process, determined from the temperature dependence of the optical transition, is 0.26 MJ (62 kcal) per mole of cooperative unit. The ratio of the two enthalpy values yields an apparent cooperative length for the A-Z transition of poly[r(G-C)] of approximately 60 base pairs, indicative of a concerted all-or-none process.     

Molten globule state of equine β-Lactoglobulin

The acid-unfolded state of equine β-lactoglobulin was characterized by means of circular dichroism, nuclear magnetic resonance, analytical gel-filtration chromatography, and analytical centrifugation. The acid-unfolded state of equine β-lactoglobulin has a substantial secondary structure as shown by the far-ultraviolet circular dichroism spectrum but lacks persistent tertiary packing of the side chains as indicated by the near-ultraviolet circular dichroism and nuclear magnetic resonance spectra. It is nearly as compact as the native conformation as shown by the gel filtration and sedimentation experiments, and it has the exposed hydrophobic surface as indicated by its tendency to aggregate. All of these characteristics indicate that the acid-unfolded state of equine β-lactoglobulin is a molten globule state. The α helix content in the acid-unfolded state, which has been estimated from the circular dichroism spectrum, is larger than that in the native state, suggesting the presence of nonnative α helices in the molten globule state. This result suggests the generality of the intermediate with nonnative α helices during the folding of proteins having the β-clam fold. © 1997 Wiley-Liss Inc.     

Importance of DNA conformation in the reaction with cis-dichlorodiammine platinum (II)

Cis-dichlorodiammine platinum (II) has been reacted with synthetic polynucleotides either in B or in Z conformation. The binding of cis-dichlorodiammine platinum (II) stabilizes the Z conformation when reacted with poly (dG-m⁵dC) ·poly (dG-m⁵dC) in the Z conformation as shown by circular dichroism and by the antibodies to Z-DNA. On the other hand, the binding of cis-dichlorodiammine platinum (II) stabilizes a new conformation when reacted with poly(dG-dC)·poly(dG-dC) or poly (dG-m⁵dC)·poly(dG-m5dC) in the B conformation. The antibodies to Z-DNA bind to these platinated polynucleotides. In rabbits, the injection of platinated poly (dG-dC) poly (dG-dC) induces the synthesis of antibodies which recognize Z-DNA. In low salt conditions, the circular dichroism spectra of these platinated polynucleotides differ from those of B-DNA or Z-DNA. The characteristic³¹P nuclear magnetic resonance spectrum of Z-DNA is not detected. It appears only at high ionic strength, as a component of a more complex spectrum.     

CD and NMR studies of prion protein (PrP) helix 1. Novel implications for its role in the PrPC-->PrPSc conversion process

The conversion of prion helix 1 from an alpha-helical into an extended conformation is generally assumed to be an essential step in the conversion of the cellular isoform PrPC of the prion protein to the pathogenic isoform PrPSc. Peptides encompassing helix 1 and flanking sequences were analyzed by nuclear magnetic resonance and circular dichroism. Our results indicate a remarkably high instrinsic helix propensity of the helix 1 region. In particular, these peptides retain significant helicity under a wide range of conditions, such as high salt, pH variation, and presence of organic co-solvents. As evidenced by a data base search, the pattern of charged residues present in helix 1 generally favors helical structures over alternative conformations. Because of its high stability against environmental changes, helix 1 is unlikely to be involved in the initial steps of the pathogenic conformational change. Our results implicate that interconversion of helix 1 is rather representing a barrier than a nucleus for the PrPC-->PrPSc conversion.     

B--Z conformational transition in d(CGCGCGTTAATT), d(CGCGCGTTAA) and d(CGCGCGTT)

Conformational studies on three DNA-oligomers (d(CGCGCGTTAATT), d(CGCGTTAA) and d(CGCGCGTT) in solution by circular dichroism spectroscopy are reported. In low salt solution, all three DNA oligomers exhibit a characteristic B-conformation. However, under the influence of high salt concentration i.e. 5M NaCl, the octamer d(CGCGCGTT) exhibits 'A' conformation whereas the decamer and dodecamer retain B-conformation. On addition of millimolar amount of NiCl2 to the 5M NaCl, solution of oligodeoxynucleotides a B-Z transition is observed in octamer, decamer and dodecamer. However, NiCl2 titrations show that mid point of transition for dodecamer is at 2.25 mM, for decamer is at 13 mM NiCl2 and for octamer is 17 mM at NiCl2. In 60% alcohol all three oligonucleotides remain in the B-conformation. The melting temperatures of oligonucleotides at various salt concentration are also reported. Thermodynamic parameters calculated by melting profile using a two state model show that dodecamer and decamer are most stable in their 5M NaCl, B-form. However, octamer is more stable in its Z form than that of its 'A' form.     

Two peptide fragments G55-I72 and K97-A109 from staphylococcal nuclease exhibit different behaviors in conformational preferences for helix formation

Two synthetic peptides, SNasealpha1 and SNasealpha2, corresponding to residues G55-I72 and K97-A109, respectively, of staphylococcal nuclease (SNase), are adopted for detecting the role of helix alpha1 (E57-A69) and helix alpha2 (M98-Q106) in the initiation of folding of SNase. The helix-forming tendencies of the two SNase peptide fragments are investigated using circular dichroism (CD) and two-dimensional (2D) nuclear magnetic resonance (NMR) methods in water and 40% trifluoroethanol (TFE) solutions. The coil-helix conformational transitions of the two peptides in the TFE-H2O mixture are different from each other. SNasealpha1 adopts a low population of localized helical conformation in water, and shows a gradual transition to helical conformation with increasing concentrations of TFE. SNasealpha2 is essentially unstructured in water, but undergoes a cooperative transition to a predominantly helical conformation at high TFE concentrations. Using the NMR data obtained in the presence of 40% TFE, an ensemble of alpha-helical structures has been calculated for both peptides in the absence of tertiary interactions. Analysis of all the experimental data available indicates that formation of ordered alpha-helical structures in the segments E57-A69 and M98-Q106 of SNase may require nonlocal interactions through transient contact with hydrophobic residues in other parts of the protein to stabilize the helical conformations in the folding. The folding of helix alpha1 is supposed to be effective in initiating protein folding. The formation of helix alpha2 depends strongly on the hydrophobic environment created in the protein folding, and is more important in the stabilization of the tertiary conformation of SNase.     

Metal binding induces conversion of B- to the hybrid B-Z-form in natural DNA

Highly polymerized herring testis DNA of the random nucleotide sequence has been studied in solution by circular dichroism and ultra-violet absorption spectrometry under various experimental conditions. At low temperature upon addition of 0.05M NaCl or 1.15M MgSO(4) the DNA formed a helix that belonged to the B-family. As the temperature was increased a transition from the pure B- to the hybrid B-Z-form occurred in the presence of 1.15M MgSO(4). This transition occurred over a large range of temperatures and corresponded to a non-cooperative conformational change. A similar DNA transition was induced with 0.098mM Co(NH(3))(6)Cl(3). However, in the presence of 5.3M NaCl the DNA conformation was not similar to that observed in 1.15M MgSO(4) or 0.098mM Co(NH(3))(6)Cl(3) independently on the environmental temperature. In 5.3M NaCl the DNA is thought to undergo a transition from one to another right-handed conformation that could be intermediate partially dehydrated conformer arising on the first step in the sequential transition to the dehydration of the polynucleotide. Our results show that a realistic model of native DNA, bearing Z-tracts embedded in B-helixes, can be obtained upon binding of alkaline earth or transition metals.     

Conformational analysis of d(C3G3), a B-family duplex in solution

NMR and circular dichroism studies of the duplex formed by the self-complementary DNA hexanucleotide d(C3G3) indicate that it is a B-type structure but differs from standard B-form. An analysis of NMR coupling constants within the deoxyribose moieties yields a 70% or greater contribution from pseudorotation phase angles corresponding to the C3'-exo conformation, a conformation similar to the C2'-endo conformation associated with B-form DNA. Intranucleotide interproton distances are consistent with a B-form structure, but some internucleotide distances are intermediate between A- and B-form structures. Circular dichroism spectra have B-form characteristics but also include an unusual negative band at 282 nm. The solution spectroscopic results are in contrast with X-ray crystallographic studies which find A-form structures for similar sequences.     

Phosphorus NMR spectra of natural DNA fragments in the course of the B-to-A conformational transition

Changes in the ³¹P-nmr spectra of sonicated natural DNA fragments were investigated in ethanol solutions where the fragments underwent, as checked by CD, the B-to-A conformational transition. The study produced the following conclusions: (1) The high DNA concentrations used for the ³¹P-nmr measurements promote the transition compared to dilute solutions that are commonly used for CD measurements. (2) The B-to-A transition was reflected in a cooperative downfield shift of the DNA ³¹P-nmr resonance, consistent with unwinding of the double helix. (3) Prior to the transition, the changes in chemical shift of double-and single-stranded DNAs were almost identical. It thus appears that the effect of ethanol on the geometry and hydration of phosphodiester linkages does not depend heavily on DNA base–base interactions. (4) The A-form resonances were 30–40% narrower than the B-form resonances, which is attributed to marked sequence-dependent variations in the latter conformation and to their reduction in the former. (5) The B-form DNA aggregated in the concentrated ³¹P-nmr samples in the presence of ethanol, judged from a milky opalescence of the solution and a substantial broadening of its ³¹P-nmr resonance. The broadening abruptly disappeared as soon as DNA adopted the A-form so that DNA, in dependence on the secondary structure, showed different tendencies to condense in the presence of ethanol. The condensation increased cooperativity of the B-to-A interconversion.     

BII nucleotides in the B and C forms of natural-sequence polymeric DNA: A new model for the C form of DNA

A combination of solid-state (31)P and (13)C NMR, X-ray diffraction, and model building is used to show that the B and C forms of fibrous macromolecular DNA consist of two distinct nucleotide conformations, which correspond closely to the BI and BII nucleotide conformations known from oligonucleotide crystals. The proportion of the BII conformation is higher in the C form than in the B form. We show structural models for a 10(1) double helix involving BI nucleotides and a 9(1) double helix involving BII nucleotides. The 10(1) BI model is similar to a previous model of B-form DNA, while the 9(1) BII model is novel. The BII model has a very deep and narrow minor groove, a shallow and wide major groove, and highly inclined bases. This work shows that the B to C transition in fibers corresponds to BI to BII conformational changes of the individual nucleotides.     

Structural analysis of 21-hydroxypregn-4-ene-3,20-dione (deoxycorticosterone) derivative compounds. Part II: Proton NMR spectra

Complete analysis of the proton nuclear magnetic resonance spectrum of desoxycorticosterone (DOC) has been made using selective double irradiation, two-dimensional experiments, relaxation rate, and nuclear Overhauser effect measurements in order to specify the structure and conformation of products encountered during the preparation of the specific antigen DOC-bovine serum albumin (BSA). It has been shown that DOC has the normal P conformation with ring A half-chair, and ring B chair. This confirms results previously obtained by circular dichroism measurements.