Conformational changes in subfractions of calf thymus histone H1.

This paper presents the first study of conformational changes in the subfractions of calf thymus H1. H1 was fractionated by the method of Kincade and Cole (Kincade, J. M., and Cole, R.D. (1966), J. Biol. Chem. 241. 5790) using a very shallow Gdn-HC1 gradient. A possible new H1 subfraction, about 5--8% of the H1, has been found and characterized by amino acid analysis and electrophoresis. The effects of salt concentration and pH on the conformation of each of the four major subfractions have been studied by measuring the fluorescence anisotropy of the tyrosine emission and the circular dichroism (CD) of the peptide bond. Upon the addition of salt to aqueous solutions at neutral pH, all four subfractions show an instantaneous change in fluorescence anisotropy, fluorescence intensity, tyrosine absorbance, and CD. The folding associated with this instantaneous change is highly cooperative, and involves the region of the molecule containing the lone tyrosine, which becomes buried in the folded form. The folding of subfraction 3a is more sensitive to salt than the other major subfractions. Upon folding, approximately 13% of the residues of subfractions 1b and 2 form alpha and beta structure; 3a and 3b have approximately 16% of the residues in alpha and beta structures. There is no evidence for interactions between the subfractions. In salt-free solutions, each of the four major subfractions show very little change in conformation in going from low to neutral pH, but each shows a very sharp transition near pH 9. This transition gives rise to a marked increase in fluorescence anisotropy and fluorescence intensity, and involves the formation of both alpha and beta strucute in a manner similar to that of the salt-induced state.     

pH effects on the structure of the inner histones

The structure of the inner histone complex extracted from chicken erythrocyte chromatin with 2 M NaCl has been studied as a function of pH. At pH 6, the complex dissociates to (H3–H4)₂ tetramer and H2A·H2B dimer, with little change in α-helix content (as monitored by circular dichroism at 222 nm). Although the circular dichroism of tyrosyl side chains is also largely unchanged by the dissociation, measurements of intrinsic fluorescence do suggest a change in the environment of one or more tyrosines as a result of dissociation. Below pH 4, the histones become partially unfolded, lose specific secondary and tertiary structure, and undergo nonspecific aggregation. Both the pH 6 and 4 transitions, which are largely reversible, parallel pH-induced structural changes of nucleosomes (Zama, M., Olins, D.E., Prescott, B. and Thomas, G.J. (1978) Nucleic Acids Res. 5, 3881–3897). The results are consistent with the presence of tyrosine residues at the histone subunit-subunit contacts and suggest that histone conformation within the globular regions is largely independent of histone-DNA interactions.     

Tyrosine fluorescence of two tryptophan-free proteins: histones H1 and H5.

The fluorescence intensity of the single tyrosine residue in histone H1 increases from RTYR = 0.3 to RTYR = 1.3 as the protein undergoes a conformational change from the random coil state to a folded form. Enhanced fluorescence in the folded state has not been observed before in ap protein. Histone H5 shows no change in fluorescence intensity on folding. This is interpreted as a result of compensation between enhanced and reduced fluorescence in the three tyrosine residues.     

Structuring of H1 histone. Evidence of high-affinity binding sites for phosphate ions

Circular dichroism studies show that low concentrations of phosphate ions induce folding of the H1 histones. Sulfate and perchlorate anions have effects similar to phosphate indicating the presence on H1 histones of binding sites with high affinity for ions with tetrahedral geometry. In fact, the structuring efficiency of different ions, as determined by the midpoint value of the effect/concentration curve, is 0.05 M for NaCl, 0.005 M for NaClO4, 0.001 M for sodium phosphate and 0.0003 M for sodium sulfate on H1 histone from Chaetopterus variopedatus sperm chromatin. Phosphate shows similar folding efficiency also on calf thymus and on sea-urchin sperm H1 histones. The effect of phosphate ions on the H1 molecule is observed also by differential absorption spectroscopy in the region of absorption of amino acid side-chains. Binding studies by gel filtration chromatography on Sephadex columns show that phosphate binding occurs in the presence of structuring concentrations of sodium chloride. About 9 ATP molecules bind to H1 histones derived from non-active cell chromatins while only 3.5 ATP molecules bind to H1 derived from active somatic chromatins. The fluorescence of the tyrosine residues of Chaetopterus sperm H1 is enhanced by chloride ions and heavily quenched by phosphate ions in correlation with structuring of the molecule, demonstrating direct interactions between tyrosine residues and phosphate ions. The defined and limited number of phosphate groups bound per histone molecule, the high affinity of the interaction and the effect on the structure of the histone suggest the participation of phosphate groups in the binding of H1 histones to DNA.     

The intrinsic tyrosine fluorescence of histone H1. Steady state and fluorescence decay studies reveal heterogeneous emission

In wavelength-resolved steady state spectra we observe three different kinds of emission from histone H1, a class A protein with only a single tyrosine residue. Unfolded H1 emissions that peak at approximately 300 and 340 nm can both be excited maximally at approximately 280 nm. Another, peaking much further to the red at approximately 400 nm, can be excited maximally at approximately 320 nm. The 300-nm fluorescence can be resolved by lifetime measurements into three components with decay times of approximately 1, 2, and 4 ns. On sodium-chloride-induced refolding of H1, simplification of the emission properties occurs. The 340 and 400-nm components disappear while the two shorter lifetime components of the 300-nm band diminish in amplitude and are replaced by the 4-ns decay. We believe that the 340-nm emission is tyrosinate fluorescence resulting from excited-state proton transfer. The origin of the 400-nm emission remains uncertain. We assign the 1 and 2-ns components of the 300-nm emission to two states of tyrosine in denatured H1 and the 4-ns decay to fluorescence of the single tyrosine residue in the globular region of refolded H1. Our results support the contention that salt induced folding of H1 is a cooperative two state process, and permit us to better understand the previously reported increases in fluorescence intensity and anisotropy on salt-induced folding.     

Correlation of tryptophan fluorescence intensity decay parameters with 1H NMR-determined rotamer conformations: [tryptophan2]oxytocin.

While the fluorescence decay kinetics of tyrosine model compounds [Laws, W. R., Ross, J. B. A., Wyssbrod, H. R., Beechem, J. M., Brand, L., & Sutherland, J. C. (1986) Biochemistry 25, 599-607] and the tyrosine residue in oxytocin [Ross, J. B. A., Laws, W. R., Buku, A., Sutherland, J. C., & Wyssbrod, H. R. (1986) Biochemistry 25, 607-612] can be explained in terms of heterogeneity derived from the three ground-state chi 1 rotamers, a similar correlation has yet to be directly observed for a tryptophan residue. In addition, the asymmetric indole ring might also lead to heterogeneity from chi 2 rotations. In this paper, the time-resolved and steady-state fluorescence properties of [tryptophan2]oxytocin at pH 3 are presented and compared with 1H NMR results. According to the unrestricted analyses of individual fluorescence decay curves taken as a function of emission wavelength and a global analysis of these decay curves for common emission wavelength-independent decay constants, only three exponential terms are required. In addition, the preexponential weighting factors (amplitudes) have the same relative relationship (weights) as the 1H NMR-determined chi 1 rotamer populations of the indole side chain. 15N was used in heteronuclear coupling experiments to confirm the rotamer assignments. Inclusion of a linked function restricting the decay amplitudes to the chi 1 rotamer populations in the individual decay curve analyses and in the global analysis confirms this correlation. According to qualitative nuclear Overhauser data, there are two chi 2 populations. Depending upon the degree of correlation between chi 2 and chi 1, there may be from three to six side-chain conformations for the tryptophan residue. The combined fluorescence and NMR results are consistent with a rotamer model in which either (i) the chi 2 rotations are fast compared to the fluorescence intensity decay of the tryptophan residue, (ii) environmental factors affecting fluorescence intensity decay properties are dominated by chi 1 interactions, or (iii) the chi 2 and chi 1 rotations are highly correlated.     

Peculiarities of the amino acid composition, spatial organization and interaction with DNA of histones H1 from calf thymus and carp spermatozoa

The amino acid composition of the H1-like histone isolated from carp spermatozoa (H1carp) is characterized by a high content of lysine (34.6%) and a low content of glycine (4.5%) as compared to that of its calf counterpart (H1calf). The Lys/Arg ratio is 21.6, which is much higher than that for the H1-like histones from other species spermatozoa (cf. echinodermata). It was shown that the fluorescence anisotropy and excitation spectra of histones H1carp and H1calf change synchronically. At the same time the final folding of the polypeptide chains of these histones within their ternary structure is different. These differences manifest themselves in a distinct quantum yield of both histones and different accessibility of the single tyrosine residue for fluorescence quenchers. In histone--DNA complexes the tyrosine fluorescence is quenched. An increase in the ionic strength gives rise to a formation of large-sized aggregates in a histone H1--DNA solution which contain structurally heterogenous histones H1 from different sources. Histone H1carp causes DNA aggregation at lower ionic strength values than its calf counterpart. The complexes are dissociated at 0.6 M NaCl.     

Synthetic peptide analogues differentially alter the binding affinities of cyclic nucleotide dependent protein kinases for nucleotide substrates

Analogues of a synthetic heptapeptide substrate corresponding to the sequence around a phosphorylation site in histone H2B [Glass, D. B. & Krebs, E. G. (1982) J. Biol. Chem. 257, 1196-1200] were used to assess interactions between the peptide substrate and the ATP binding sites of cGMP-dependent protein kinase and the catalytic subunit of cAMP-dependent protein kinase. The affinity of each protein kinase for lin-benzo-ADP was determined in the absence and presence of substrate peptide by fluorescence anisotropy titrations [Bhatnagar, D., Roskoski, R., Jr., Rosendahl, M. S., & Leonard, N. J. (1983) Biochemistry 22, 6310-6317]. The Kd values of cGMP-dependent protein kinase for lin-benzo-ADP in the absence and presence of cGMP were 7.6 and 9.7 microM, respectively. Histone H2B(29-35) (Arg-Lys-Arg-Ser-Arg-Lys-Glu) had no effect on nucleotide affinity in either the absence or presence of cGMP. However, when lysine-34 located two residues after the phosphorylatable serine is replaced with an alanyl residue, the resulting [Ala34]histone H2B(29-35) and its analogue peptides interact with cGMP-dependent protein kinase and/or the nucleotide in a fashion that decreases nucleotide binding affinity approximately 3-fold. This amino acid replacement had previously been shown to cause an increase in Vmax and a decrease in the pH optimum for the phosphotransferase reaction. Replacement of positively charged residues at positions 30 and 31 of the peptide also decreased nucleotide affinity. Other analogues of histone H2B(29-35) failed to affect binding of lin-benzo-ADP to the active site of the cGMP-dependent enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescence study of the effects of histone tyrosyl residues of DNA

The fluorescence spectra of histone-DNA complexes at pH 7.0 were studied. These species showed fluorescence spectra broader than the characteristic histone spectrum, with the appearance of peaks at higher wavelengths. It is shown that the emission at higher wavelengths arises from the bases of DNA (specially from the A-T bases), when their hydrogen bonds are weakened or broken. These results corroborate those of other authors and it is postulated that tyrosine residues in histone form hydrogen bonds with DNA and/or stack with the bases of the nucleic acid.     

Histone H1 structure probed by Staphylococcus aureus V8-proteinase

Proteolytic digestion of calf thymus histone H1 with Staphylococcus aureus V8-proteinase under structuring conditions generates one major limit peptide P1 which consists of approx. 170 residues. Edman degradation establishes the N-terminal sequence as: Leu-Ile-Thr-Lys-Ala-Val-Ala-Ala-Ser-Lys. Chymotryptic fingerprinting shows that the C-terminal part of the H1 molecule is fully preserved. The peptide therefore comprises the residues H1 (42-210). The Glu-41 cleavage is extremely unusual as it occurs in the structured G-domain which is known to be resistant to proteinases (Hartman, P. G., Chapman, G. E., Moss, T. and Bradbury, E. M. (1977) Eur. J. Biochem. 77, 45-71; B?hm, L., Sautière, P., Cary, P. D. and Crane-Robinson, C. (1982) Biochem. J. 203, 577-582). The V8-proteinase cleavage product H1 (42-210) shows only 20% folding as compared to 95-99% folding shown by the peptides H1 (34-121), H1 (31-210) and H1 (33-210). Folding of the G-domain thus critically depends upon the presence of the eight residues 33-41 amongst which the Gly-Pro-Pro sequence at position 36-38 and a beta-turn predicted at position 35 are considered to be particularly important. The location of the cleavage site in the G-domain renders Staphylococcus aureus V8-proteinase suitable as a structural probe.     

Extracellular domains,transmembrane segments,and intracellular domains interact to determine the cation selectivity of Na,K- and gastric H,K-ATPase

We have previously reported that three residues of the fourth transmembrane segment (TM4) of the Na,K- and gastric H,K-ATPase alpha-subunits appear to play a major role in the distinct cation selectivities of these pumps [Mense, M., et al. (2000) J. Biol. Chem. 275, 1749-1756]. Substituting these three residues in the Na,K-ATPase sequence with their H,K-ATPase counterparts (L319F, N326Y, T340S) and replacing the TM3-TM4 ectodomain sequence with that of the H,K-ATPase alpha-subunit result in a pump that exhibits 50% of its maximal ATPase activity in the absence of Na(+) when the assay is performed at pH 6.0. This effect is not seen when the ectodomain alone is replaced. To gain more insight into the contributions of the three residues to establishing the selectivity of these pumps for Na(+) ions versus protons, we generated Na,K-ATPase constructs in which these residues are replaced by their H,K-ATPase counterparts either singly or in combinations. Surprisingly, none of the point mutants nor even the triple mutant was able to hydrolyze ATP at pH 6.0 at a rate greater than 20% of their respective V(max)s. For the point mutants L319F and N326Y, protons seem to competitively inhibit ATP hydrolysis at pH 6.0, based on the low apparent affinity for Na(+) ions at pH 6.0 compared to pH 7.5. It would appear, therefore, that the cation selectivity of Na,K- and H,K-ATPase is generated through a cooperative effort between residues of transmembrane segments and the flanking loops that connect these transmembrane domains. This view is further supported by homology modeling of the Na,K-ATPase based on the crystal structure of the SERCA pump.     

Accessibility of histone oligomers to the action of trypsin in a solution or in chromatin with different degrees of compactness

The accessibility to trypsin of "core" histones within the dimer (H2A-H2B), tetramer (H3-H4)2, octamer (H2A-H2B-H3-H4)2 and in chromatin was studied. It was shown that the hydrolysis of histones H2A and H2B within the dimer and octamer occurs in essentially the same way. The tetramer (H2-H4)2 becomes more compact with an increase in the ionic strength. Some of the tetramer (H3-H4)2 sites within the octamer are protected against trypsin. It was demonstrated that in terms of the histone accessibility to trypsin chromatin can exist in three states, i.e., tightly packed (in the presence of histone H1 and bivalent cations), intermediate (in the absence of histone H1 or bivalent cations) and folded (in the absence of histone H1 and bivalent cations). The folding of histones in neither of these chromatin states coincides with that within the octamer in 2M NaCl.     

pH-dependent stability and folding kinetics of a protein with an unusual alpha-beta topology: the C-terminal domain of the ribosomal protein L9

The folding kinetics and thermodynamics of the isolated C-terminal domain of the ribosomal protein L9 (CTL9) have been studied as a function of pH. CTL9 is an alpha-beta protein that contains a single beta-sheet with an unusual mixed parallel, anti-parallel topology. The folding is fully reversible and two-state over the entire pH range. Stopped-flow fluorescence and CD experiments yield the same folding rate, and the chevron plots have the characteristic V-shape expected for two-state folding. The values of DeltaG*(H2O) and the m value calculated from the kinetic experiments are in excellent agreement with the equilibrium measurements. The extrapolated initial amplitudes of both the stopped-flow fluorescence and CD measurements show that there is no detectable burst phase intermediate. The domain contains three histidine residues, two of which are largely buried in the native state. They do not participate in salt-bridges or take part in a hydrogen bonded network. NMR measurements reveal that the buried histidine residues have significantly perturbed pK(a) values in the native state. The equilibrium stability and the folding rate are found to be strongly dependent upon their ionization state. There is a linear relationship between the log of the folding rate and DeltaG* (H2O) . The protein is much more stable and folds noticeably faster at pH values above the native state pK(a) values. DeltaG*(H2O) of unfolding increases from 2.90 kcal mol(-1) at pH 5.0 to 6.40 kcal mol(-1) at pH 8.0 while the folding rate increases from 0.60 to 18.7 s(-1). Tanford linkage analysis revealed that the interactions involving the two histidine residues are largely developed in the transition state. The results are compared to other studies of the pH-dependence of folding.     

Spatial organization of the (H3-H4-H2A-H2B)2 histone octamer

Structure of the (H2A-H2B-H3-H4)2 histone octamer isolated from calf thymus chromatin at ionic strength 0.1 to 4.0 M NaCl, pH 7.6, was studied spectrofluorometrically. Sensitivity of lambda max tyrosine fluorescence position to structural changes of histone oligomers and to the processes of their association was shown. It were detect two ranges of cooperative changes in histone optical parameters at 0.6-1.4 M NaCl (transition I) and at 2.4-3.4 M NaCl (transition II): Transition I corresponds to the formation of equilibrium system (hexamer) + (dimer) in equilibrium octamer. Transition II corresponds to the structural changes of the histone octamer. Thus, fluorescence anisotropy increases, lambda max for fluorescence spectrum is shifted to the longer wavelengths, contributions of two components to fluorescence decay change, a fraction of fluorescence accessible to the quenching by I- decreases. Histone octamer formation is characterized by making specific contacts between the (H2A-H2B) dimer and (H3-H4)2 tetramer. These contacts are realized at gradual changing of ionic strengths (by dialysis). In the case of abrupt local changes of the environment the process is irreversibly shifted to formation of unspecific high molecular aggregates. The important function role for energetically degenerated states of histone oligomers, energy barriers between which can be overcome by changing total conditions of histone microenvironment in chromatin is discussed.     

Role of individual histone tyrosines in the formation of the nucleosome complex.

We have determined the accessibility of histone tyrosine residues to react with p-nitrobenzenesulfonyl fluoride (NBSF) in intact nuclei, salt-dissociated nucleosomes, isolated histone complexes, and individual core histones. Of the 15 core histone tyrosine residues, 13 are inaccessible in native nucleosomes; only Tyr121 near the C-terminus of H2B is fully accessible, and Tyr54 of H3 is partially accessible under near-physiological conditions. When H1 and the basic N-terminal tails of the core histones are dissociated from the DNA by treating nuclei with 0.4 and 0.8 M NaCl, the two tyrosines which are adjacent to the basic regions of H2B and H3 become accessible as well. This indicates that these tyrosine residues may be involved in histone-DNA interactions, either directly or indirectly. When the H2A-H2B dimers are dissociated from the chromatin by raising the NaCl concentration to 1.2 M, three to four tyrosines located in the structured regions of H2B and H4 are exposed, suggesting that these tyrosine residues may be located at the dimer-tetramer interface. Dissociating all the histones from the DNA at an even higher ionic strength as a mixture of dimers, tetramers, and octamers does not change the pattern of Tyr exposure, but reduces the reactivity of the tyrosines at the dimer-tetramer interface as would be expected from the reassociation of H2A-H2B dimers and H3-H4 tetramers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of the histone tetramer (H3-H4)2: 2. Position of λmax in the tyrosyl fluorescence spectra and tyrosyl accessibility to quenchers

It was shown that the histone tetramer (H3-H4)₂ fluorescence spectra were shifted by about 2 nm towards the long-wave region and had a larger halfwidth than the free tyrosine fluorescence spectra. Denaturation with 8 m urea resulted in a shift towards the short-wave region and a decrease in the halfwidth of the histone tetramer (H3-H4)₂ tyrosine fluorescence spectra. Fluorescence quenching of the histone tetramer (H3-H4)₂ by iodine ions was analysed by the Stern-Volmer equation. It was estimated that at 0.1 m NaCl and 0.3–0.8 m NaCl, 45% and 60% tyrosyl fluorescence, respectively, was quenched by I^? ions. The results obtained suggests that histone tetramer (H3-H4)₂ may have several structural forms distinguished by the amount of ‘exposed’ and ‘buried’ tyrosyls depending upon the conditions of the medium.     

The mechanism of nucleosome assembly onto oligomers of the sea urchin 5 S DNA positioning sequence

We have used a model system composed of tandem repeats of Lytechinus variegatus 5 S rDNA (Simpson, R. T., Thoma, F., and Brubaker, J. M. (1985) Cell 42, 799-808) reconstituted into chromatin with chicken erythrocyte core histones to investigate the mechanism of chromatin assembly. Nucleosomes are assembled onto the DNA template by mixing histone octamers and DNA in 2 M NaCl followed by stepwise dialysis into very low ionic strength buffer over a 24-h period. By 1.0 M NaCl, a defined intermediate composed of arrays of H3.H4 tetramers has formed, as shown by analytical and preparative ultracentrifugation. Digestion with methidium propyl EDTA.Fe(II) indicates that these tetramers are spaced at 207 base pair intervals, i.e. one/repeat length of the DNA positioning sequence. In 0.8 M NaCl, some H2A.H2B has become associated with the H3.H4 tetramers and DNA. Surprisingly, under these conditions DNA is protected from methidium propyl EDTA.Fe(II) digestion almost as well as in the complete nucleosome, even though these structures are quite deficient in H2A.H2B. By 0.6 M NaCl, nucleosome assembly is complete, and the MPE digestion pattern is indistinguishable from that observed for oligonucleosomes at very low ionic strength. Below 0.6 M NaCl, the oligonucleosomes are involved in various salt-dependent conformational equilibria: at approximately 0.6 M, a 15% reduction in S20,w that mimics a conformational change observed previously with nucleosome core particles; at and above 0.1 M, folding into a more compact structure(s); at and above 0.1 M NaCl, a reaction involving varying amounts of dissociation of histone octamers from a small fraction of the DNA templates. In low ionic strength buffer (less than 1 mM NaCl), oligonucleosomes are present as fully loaded templates in the extended beads-on-a-string structure.     

A comparison of the intrinsic fluorescence of red kangaroo, horse and sperm whale metmyoglobins

Several metmyoglobins (red kangaroo, horse and sperm whale), containing different numbers of tyrosines, but with invariant tryptophan residues (Trp-7, Trp-14), exhibit intrinsic fluorescence when studied by steady-state front-face fluorometry. The increasing tyrosine content of these myoglobins correlates with a shift in emission maximum to shorter wavelengths with excitation at 280 nm: red kangaroo (Tyr-146) emission maximum 335 nm; horse (Tyr-103, -146) emission maximum 333 nm; sperm whale (Tyr-103, -146, -151) emission maximum 331 nm. Since 280 nm excites both tyrosine and tryptophan, this strongly suggests that tyrosine emission is not completely quenched but also contributes to this fluorescence emission. Upon titration to pH 12.5, there is a reversible shift of the emission maximum to longer wavelengths with an increase greater than 2-fold in fluorescence intensity. With excitation at 305 nm, a tyrosinate-like emission is detected at a pH greater than 12. These studies show that: (1) metmyoglobins, Class B proteins containing both tyrosine and tryptophan residues, exhibit intrinsic fluorescence; (2) tyrosine residues also contribute to the observed steady-state fluorescence emission when excited by light at 280 nm; (3) the ionization of Tyr-146 is likely coupled to protein unfolding.     

Unusual protonated structure in the homopurine.homopyrimidine tract of supercoiled and linearized plasmids recognized by chemical probes

Plasmid pEJ4, which is a derivative of pUC19 containing an insert with 60-bp-long homopurine.homopyrimidine tract from sea urchin P. miliaris histone gene spacer, was studied by chemical probes of the DNA structure osmium tetroxide and glyoxal. The former probe reacts with pyrimidine bases, while the latter forms a stable product only with guanine residues. These probes can thus be applied as specific probes for the homopyrimidine and homopurine strands, respectively. At pH 6.0 the site-specific modification of the homopurine.homopyrimidine tract by both probes was observed at native superhelical density of the plasmid. In the linear plasmid under the same conditions this modification was absent; it appeared, however, at more acid pH values. In supercoiled DNA the hypersensitivity of the homopurine.homopyrimidine tract to osmium tetroxide did not substantially change when pH was decreased from 6.0 to 4.0. Changes in NaCl concentration at pH 4.5 did not influence the hypersensitivity to osmium tetroxide; at pH 6.0 this hypersensitivity decreased with increasing NaCl concentration. These results thus show that the chemical probes recognize an unusual protonated structure containing unpaired bases or non-Watson-Crick base pairs. At pH 5.6 the site-specific modification occurred at or near to the middle of the homopurine.homopyrimidine tract, suggesting that a hairpin may be involved in the unusual structure under the given conditions. From the models suggested so far for the unusual structure of homopurine.homopyrimidine tracts our results fit best the protonated triplex H form suggest by V.I. Lyamichev, S.M. Mirkin and M.D. Frank-Kamenetskii, J. Biomol. Struct. Dyn. 3,667 (1986).