The formation of adjacent triplex-duplex domainsin DNA.

The ability of single-stranded DNA oligomers to form adjacent triplex and duplex domains with two DNA structural motifs was examined. Helix-coil transition curves and a gel mobility shift assay were used to characterize the interaction of single-stranded oligomers 12-20 nt in length with a DNA hairpin and with a DNA duplex that has a dangling end. The 12 nt on the 5'-ends of the oligomers could form a triplex structure with the 12 bp stem of the hairpin or the duplex portion of the DNA with a dangling end. The 3'-ends of the 17-20 nt strands could form Watson-Crick pairs to the five base loop of the hairpin or the dangling end of the duplex. Complexes of the hairpin DNA with the single-stranded oligomers showed two step transitions consistent with unwinding of the triplex strand followed by hairpin denaturation. Melting curve and gel competition results indicated that the complex of the hairpin and the 12 nt oligomer was more stable than the complexes involving the extended single strands. In contrast, results indicated that the extended single-stranded oligomers formed Watson-Crick base pairs with the dangling end of the duplex DNA and enhanced the stability of the adjacent triplex region.     

NMR study of a synthetic DNA hairpin

The secondary structure of the synthetic oligodeoxyribonucleotide d(CGCGCGTTTTCGCGCG) (I) has been demonstrated to be a unimolecular hairpin structure (hairpin I) over a wide range of oligonucleotide concentrations (2 X 10(-5) to 1.6 X 10(-3) M) and temperature (0-87 degrees C). The assignments of the resonances to specific protons were carried out by use of two-dimensional nuclear Overhauser effect and COSY spectra and by comparison with the spectra of the duplex formed by d(CG)3. Comparison of hairpin I and the hairpin of d(ATCCTATTTTTAGGAT) (II) reveals that the exchange of imino protons in stem base pairs with solvent is much slower in I than in II. However, the exchange of thymine imino protons in the loop region is much faster in I than in II even though both hairpins contain four unpaired thymine residues. The secondary structure of hairpin I contains only six G X C base pairs, yet it is more stable than the d(CG)8 duplex containing 16 G X C base pairs at all concentrations of duplex lower than 10(-3) M. These observations suggest that intramolecular hairpin formation may effectively compete with bimolecular duplex formations when the appropriate intramolecular base pairs can form.     

Solution conformation of an RNA hairpin loop.

The hairpin conformation adopted by the RNA sequence 5'GCGAUUUCUGACCGCC3' has been studied by one- and two-dimensional NMR spectroscopy. Exchangeable imino spectra in 60 mM Na+ indicate that the hairpin has a stem of six base pairs (indicated by boldface type) and a loop of three nucleotides. NOESY spectra of nonexchangeable protons confirm the formation of the stem region. The duplex has an A-conformation and contains an A.C apposition; a G.U base pair closes the loop region. The stem nucleotides have C3'-endo sugar conformations, as expected of an A-form duplex, whereas the three loop nucleotides adopt C2'-endo sugar puckers. Stacking within the loop, C8 upon the sugar of U7, stabilizes the structure. The pH dependence of both the exchangeable and nonexchangeable NMR spectra is consistent with the formation of an A+.C base pair, protonated at the N1 position of adenine. The stability of the hairpin was probed by using absorbance melting curves. The hairpin structure with the A+.C base pair is about +2 kcal/mol less stable in free energy at 37 degrees C than the hairpin formed with an A.U pair replacing the A+.C pair.     

Targeting degradation of RNA by RNase H using DNA hairpins

RNase H degradation of two 15 nt RNA target sites was examined in the presence of hairpin DNAs with a 5 nt loop and a 10 bp stem or single-stranded 15 nt DNAs. One target site was a segment of a 79 nt RNA, and the other was part of a 53 nt RNA. Secondary structure predictions indicate that the 53 nt RNA target site is entirely single stranded, while a portion of the 79 nt RNA target site forms an intramolecular duplex. Less RNase H and DNA were needed to cleave the 53 nt RNA target site than the less accessible 79 nt RNA site. The hairpin DNAs had their 5 nt loop and 3' side of the stem fully complementary to the target sites or had sequence changes that produced one to nine mismatched pairs. T(m) values ranged from 57 to 80 degrees C. The stability of the hairpin DNAs relative to the stability of their corresponding RNA-DNA hybrids influenced the extent of RNase H degradation at 37 degrees C. Under the assay conditions employed, the amount of degradation directed by the hairpin DNAs was correlated with their predicted DeltaG(o) (37) of binding to the RNA targets. A DNA hairpin with one mismatch to the target site of the 79 nt RNA did not induce degradation under conditions where fully complementary DNA hairpins produced 50-80% degradation. The in vitro results indicate that DNA hairpins can enhance the stringency of RNase H targeted degradation of the RNA sites.     

A parallel stranded linear DNA duplex incorporating dG.dC base pairs

DNA oligonucleotides with appropriately designed complementary sequences can form a duplex in which the two strands are paired in a parallel orientation and not in the conventional antiparallel double helix of B-DNA. All parallel stranded (ps) molecules reported to date have consisted exclusively of dA.dT base pairs. We have substituted four dA.dT base pairs of a 25-nt parallel stranded linear duplex (ps-D1.D2) with dG.dC base pairs. The two strands still adopt a duplex structure with the characteristic spectroscopic properties of the ps conformation but with a reduced thermodynamic stability. Thus, the melting temperature of the ps duplex with four dG.dC base pairs (ps-D5.D6) is 10-16 degrees C lower and the van't Hoff enthalpy difference delta HvH for the helix-coil transition is reduced by 20% (in NaCl) and 10% (in MgCl2) compared to that of ps-D1.D2. Based on energy minimizations of a ps-[d(T5GA5).d(A5CT5)] duplex using force field calculations we propose a model for the conformation of a trans dG.dC base pair in a ps helix.     

Unusual DNA duplex and hairpin motifs

Single-stranded DNA or double-stranded DNA has the potential to adopt a wide variety of unusual duplex and hairpin motifs in the presence (trans) or absence (cis) of ligands. Several principles for the formation of those unusual structures have been established through the observation of a number of recurring structural motifs associated with different sequences. These include: (i) internal loops of consecutive mismatches can occur in a B-DNA duplex when sheared base pairs are adjacent to each other to confer extensive cross- and intra-strand base stacking; (ii) interdigitated (zipper-like) duplex structures form instead when sheared G·A base pairs are separated by one or two pairs of purine·purine mismatches; (iii) stacking is not restricted to base, deoxyribose also exhibits the potential to do so; (iv) canonical G·C or A·T base pairs are flexible enough to exhibit considerable changes from the regular H-bonded conformation. The paired bases become stacked when bracketed by sheared G·A base pairs, or become extruded out and perpendicular to their neighboring bases in the presence of interacting drugs; (v) the purine-rich and pyrimidine-rich loop structures are notably different in nature. The purine-rich loops form compact triloop structures closed by a sheared G·A, A·A, A·C or sheared-like G_anti·C_syn base pair that is stacked by a single residue. On the other hand, the pyrimidine-rich loops with a thymidine in the first position exhibit no base pairing but are characterized by the folding of the thymidine residue into the minor groove to form a compact loop structure. Identification of such diverse duplex or hairpin motifs greatly enlarges the repertoire for unusual DNA structural formation.     

Energetics of the hairpin to mismatched duplex transition of d(GCCGCAGC) on NaCl solution

We report Potential of Mean Force studies to describe the relative thermodynamic stabilities of d(GCCGCAGC) in a mismatched duplex and a hairpin monomer conformation in NaCl solution. The PMF calculations are combined with previous molecular mechanics and normal mode analysis in order to estimate the role of different components of the free energy in determining the relative stability of the duplex and hairpin structures. The high entropy associated with the loop region and the lack of minor groove phosphate-phosphate interactions in the hairpin compete against the gain in enthalpic contribution to the free energy due to base pairing in the mismatched duplex. The combined free energy calculations show that the hairpin is the most stable conformation at low salt and that a hairpin to duplex transition takes place at approximately 0.47 M NaCl. In addition, we studied the hairpin to partially stacked single helical conformation equilibrium at low salt. We found a small variation in transition temperature in salt concentration, delta Tm/delta log10(cs) approximately 2-3 degrees K/decade, in contrast to the duplex to hairpin or duplex to partially stacked single helix transition where the transition temperature exhibited marked dependence on salt concentration. This is in qualitative agreement with experimental data. Based on the Potential of Mean Force free energy calculation, the order of relative stability of the three-conformations studied varies with salt concentration. We observed the following orders of stability: stacked single helix greater than hairpin greater than duplex for cs less than 0.77 M NaCl; single helix greater than duplex greater than hairpin for 0.77 less than Cs less than 2.1 M; and duplex greater than hairpin greater than single strand for cs greater than 2.1 M. From the calculated PMF free energy curves in the NaCl concentration range, 0.012 less than cs less than 5.0 M, we can assign upper and lower bounds for the non-ionic differences in free energy between the duplex, hairpin, and stacked single helical states (at standard conditions: cs = 1.0 M, T = 25 degrees C, and 1 M oligomer concentration). We found that for delta G duplex single helix = G duplex - 2 x G single helix less than -7.38 Kcal/mol, the single helix is the least stable state. For the duplex-to-hairpin free energy difference in the range, -1.87 less than delta G duplex-hairpin less than 0.03 Kcal/mol, there will always be a salt-induced hairpin-to-duplex transition for 0.01 less than cs less than 1.6 M NaCl. If delta G duplex-hairpin less than -1.87, the duplex is always more stable than the hairpin; and for delta G duplex-hairpin greater than Kcal/mol, the hairpin state is always more stable than the duplex, for all salt concentrations.     

Non-nearest-neighbor dependence of stability for group III RNA single nucleotide bulge loops

Thirty-five RNA duplexes containing single nucleotide bulge loops were optically melted and the thermodynamic parameters for each duplex determined. The bulge loops were of the group III variety, where the bulged nucleotide is either a AG/U or CU/G, leading to ambiguity to the exact position and identity of the bulge. All possible group III bulge loops with Watson–Crick nearest-neighbors were examined. The data were used to develop a model to predict the free energy of an RNA duplex containing a group III single nucleotide bulge loop. The destabilization of the duplex by the group III bulge could be modeled so that the bulge nucleotide leads to the formation of the Watson–Crick base pair rather than the wobble base pair. The destabilization of an RNA duplex caused by the insertion of a group III bulge is primarily dependent upon non-nearest-neighbor interactions and was shown to be dependent upon the stability of second least stable stem of the duplex. In-line structure probing of group III bulge loops embedded in a hairpin indicated that the bulged nucleotide is the one positioned further from the hairpin loop irrespective of whether the resulting stem formed a Watson–Crick or wobble base pair. Fourteen RNA hairpins containing group III bulge loops, either 3′ or 5′ of the hairpin loop, were optically melted and the thermodynamic parameters determined. The model developed to predict the influence of group III bulge loops on the stability of duplex formation was extended to predict the influence of bulge loops on hairpin stability.     

A Parallel Stranded Linear DNA Duplex Incorporating dG · dC Base Pairs

Abstract

DNA oligonucleotides with appropriately designed complementary sequences can form a duplex in which the two strands are paired in a parallel orientation and not in the conventional antiparallel double helix of B-DNA. All parallel stranded (ps) molecules reported to date have consisted exclusively of dA · dT base pairs. We have substituted four dA · dT base pairs of a 25-nt parallel stranded linear duplex (ps-D1 · D2) with dG · dC base pairs. The two strands still adopt a duplex structure with the characteristic spectroscopic properties of the ps conformation but with a reduced thermodynamic stability. Thus, the melting temperature of the ps duplex with four dG · dC base pairs (ps-D5 · D6) is 10-16°C lower and the van't Hoff enthalpy difference Δ_vH for the helix-coil transition is reduced by 20% (in NaCl) and 10% (in MgCl₂) compared to that of ps-Dl · D2. Based on energy minimizations of a ps-[d(T₅GA₅) · d(A₅CT₅)] duplex using force field calculations we propose a model for the conformation of a trans dG · dC base pair in a ps helix.     

The solution structure of a DNA hairpin containing a loop of three thymidines determined by nuclear magnetic resonance and molecular mechanics.

We have determined by two-dimensional nuclear magnetic resonance studies and molecular mechanics calculations the three-dimensional solution structure of a 21 residue oligonucleotide capable of forming a hairpin structure with a loop of three thymidine residues. This structure is in equilibrium with a duplex form. At 33 degrees C, low ionic strength and in the presence of MgCl2 the hairpin form dominates in solution. Six Watson-Crick base pairs are formed topped by the loop structure. The residues 1-3 and 18-21 are not complementary and form dangling ends. Distance constraints have been derived from nuclear Overhauser enhancement measurements. These, together with molecular mechanics calculations, have been used to determine the structure. We do not observe stacking of thymidine residues either over the 3' or the 5' end of the stem.     

Evidence for a DNA triplex in a recombination-like motif: I. Recognition of Watson-Crick base pairs by natural bases in a high-stability triplex

Data are presented on a triplex type with two parallel homologous strands for which triplex formation is almost as strong as duplex formation at least for some sequences and even at pH 7 and 0.2 M NaCl. The evidence mainly rests upon comparing thermodynamic properties of similar systems. A paperclip oligonucleotide d(A12C4T12C4A12) with two linkers C4 obviously can form a triplex with parallel back-folded adenine strand regions, because the single melting transition of this complex splits in two transitions by introducing mismatches only in the third strand region. Respectively, a hairpin duplex d(A12C4T12) and a single strand d(A12) form a triplex as a 1:1 complex in which the second adenine strand is parallel oriented to the homologous one in the Watson-Crick paired duplex. In this system the melting temperature T(m) of the triplex is practically the same as that of the duplex d(A12)-d(T12), at least within a complex concentration range of 0.2-4.0 microM. The melting behaviour of complexes between triplex stabilizing ligand BePI and the system hairpin duplex plus single strand supports the triplex model. Non-denaturing gel electrophoresis suggests the existence of a triplex for a system in which five of the twelve A-T*A base triads are substituted by C-G*C base triads. The recognition between any substituted Watson-Crick base pair (X-Y) in the hairpin duplex d(A4XA7C4T7YT4) and the correspondingly replaced base (Z) in the third strand d(A4ZA7) is mutually selective. All triplexes with matching base substitutions (Z = X) have nearly the same stability (T(m) values from 29 to 33.5 degrees C), whereas triplexes with non-matching substitutions (Z not equal X) show a clearly reduced stability (T(m) values from 15 to 22 degrees C) at 2microM equimolar oligonucleotide concentration. Most nucleic acid triple helices hitherto known are limited to homopurine-homopyrimidine sequences in the target duplex. A stable triplex formation is demonstrated for inhomogeneous sequences tolerating at least 50% pyrimidine content in the homologous strands. On the basis of the surprisingly similar thermodynamic parameters for duplex and triplex, and of the fact that this triplex type seems to be more stable than many other natural DNA triplexes known, and on the basis of semiempirical and molecule mechanical calculations, we postulate bridging interactions of the third strand with the two other strands in the triplex according to the recombination motif. This triplex, denoted by us 'recombination-like form', tolerates heterogeneous base sequences.     

NMR solution structure of the L 9.1a region of Tetrahymena group I intron.

The solution structure of 20 mer RNA contained of the loop 9.1a region of Tetrahymena group I intron was studied by NMR. This RNA oligomer has hairpin and duplex structures at high concentration (1 mM) of the sample even at low NaCl concentration (5 mM). In the hairpin structure, GC base pairs by the loop-loop interaction are formed. As study of NOESY measurements, and by the compared with the sequence, this loop region is presumed to interact with the loop 5c.     

Effect of base-pair sequence on the conformations and thermally induced transitions in oligodeoxyribonucleotides containing only AT base pairs

Tm curves, CD spectra, and kinetics results of the self-complementary DNA dodecamers d(A6T6), d(A3T3A3T3), d(A2T2A2T2A2T2), d(ATATATATATAT), and d(T6A6) demonstrate that the thermal transitions of these oligomers at low salt concentration involve a hairpin intermediate. At high salt concentrations (greater than 0.1 M Na+) only a duplex to denatured-strand transition appears to occur. The temperature and salt-concentration regions of the transitions are very sequence dependent. Alternating-type AT sequences have a lower duplex stability and a greater tendency to form hairpins than sequences containing more nonalternating AT base pairs. Of the two nonalternating sequences, d(T6A6) is significantly less stable than d(A6T6). Both oligomers have CD curves that are very similar to the unusual CD spectrum of poly(dA).poly(dT). The Raman spectra of these two oligomers are also quite similar, but at low temperature, small intensity differences in two backbone modes and three nucleoside vibrations are obtained. The hairpin to duplex transition for the AT dodecamers was examined by salt-jump kinetics measurements. The transition is faster than transitions for palindromic-sequence oligomers containing terminal GC base pairs. Stopped-flow kinetics studies indicate that the transition is second order and has a relatively low activation energy. The reaction rate increases with increasing ionic strength. These results are consistent with a three-step mechanism for the hairpin to duplex reaction: (i) fraying of the hairpin oligomers' terminal base pairs, (ii) a rate-determining bimolecular step involving formation of a cruciform-type intermediate from two hairpin oligomers with open terminal base pairs, and (iii) base-pair migration and formation in the intermediate to give the duplex.     

Use of ultra stable UNCG tetraloop hairpins to fold RNA structures: thermodynamic and spectroscopic applications.

RNA molecules of > 20 nucleotides have been the focus of numerous recent NMR structural studies. Several investigators have used the UNCG family of hairpins to ensure proper folding. We show that th UUCG hairpin has a minimum requirement of a two base-pair stem. Hairpins with a CG loop closing base pair and an initial 5'CG or 5'GC base pair have a melting temperature approximately 55 degrees C in 10 mM sodium phosphate. The high stability of even such small hairpins suggests that the hairpin can serve as a nucleation site for folding. For high resolution NMR work, the UNCG loop family (UACG in particular) provides excellent spectroscopic markers in one-dimensional exchangeable spectra, in two-dimensional COSY spectra and in NOESY spectra that clearly define it as forming a hairpin. This allows straightforward initiation of chemical shift assignments.     

Role of the closing base pair for d(GCA) hairpin stability: free energy analysis and folding simulations

Hairpin loops belong to the most important structural motifs in folded nucleic acids. The d(GNA) sequence in DNA can form very stable trinucleotide hairpin loops depending, however, strongly on the closing base pair. Replica-exchange molecular dynamics (REMD) were employed to study hairpin folding of two DNA sequences, d(gcGCAgc) and d(cgGCAcg), with the same central loop motif but different closing base pairs starting from single-stranded structures. In both cases, conformations of the most populated conformational cluster at the lowest temperature showed close agreement with available experimental structures. For the loop sequence with the less stable G:C closing base pair, an alternative loop topology accumulated as second most populated conformational state indicating a possible loop structural heterogeneity. Comparative-free energy simulations on induced loop unfolding indicated higher stability of the loop with a C:G closing base pair by ~3 kcal mol(-1) (compared to a G:C closing base pair) in very good agreement with experiment. The comparative energetic analysis of sampled unfolded, intermediate and folded conformational states identified electrostatic and packing interactions as the main contributions to the closing base pair dependence of the d(GCA) loop stability.     

Oligodeoxynucleotide folding in solution: loop size and stability of B-hairpins

The secondary structures of the synthetic DNA fragments d(CGCGCGTTTTTCGCGCG) (T5), d(CGCGCGAAAAACGCGCG) (A5), d(CGCGCGTACGCGCG) (TA), and d(CGCGCGATCGCGCG) (AT) were investigated in a combined electrophoretic and spectroscopic study. All the oligomers exist, at low temperature and over a wide range of ionic strength (0.5-100 mM salt) and of nucleotide concentration [0.1-2.0 mM (phosphate)], as a mixture of two slowly interconverting species, identified as the dimeric duplex and the monomeric hairpin structure. The thermodynamic parameters for hairpin denaturation of T5, A5, TA, and AT and for duplex denaturation of d(CGCGCG) show that (a) the hairpins are more stable than the reference hexamer duplex at all accessible nucleotide concentrations; (b) the loop contributes favorably to the enthalpy change of hairpin denaturation in the four DNA fragments; (c) the base composition of the loop (A vs T) and the size of the loop (A5/T5 vs TA/AT) do not appreciably influence the enthalpic contents of the hairpins; (d) hairpins TA and AT, with two AT bases intervening in the CG self-complementary part of the molecule, exhibit a markedly higher thermal stability than hairpins T5 and A5, which is entropic in origin. These findings are consistent with the presence of two-residue loops in the tetradecamers TA and AT.     

Unusual duplex formation in purine rich oligodeoxyribonucleotides

The purine rich oligodeoxyribonucleotides 1C, d(ATGACGGAATA) and 2C, d(ATGAGCGAATA) alone exhibit highly cooperative melting transitions. Analysis of the concentration dependence of melting, and electrophoretic studies indicate that these oligomers can form an unusual purine rich offset double helix. The unusual duplex is predicted to contain four A.T, two G.C, and four G.A mismatch base pairs as well as a single A base stacked on the 3' end of each chain of the helix. Other possible models for the duplex are unlikely because they are predicted to contain many base pairs of low stability. Changing the central sequence to CGG or GGG should destabilize the duplex and this is observed. The unusual duplex of 2C is more stable than the duplex of 1C indicating that the stability of G.A base pairs is quite sensitive to the surrounding sequence. Addition of 1C and 2C to their complementary pyrimidine strands results in normal duplexes of similar stability. We feel that the unusual duplexes are significantly stabilized by the intrinsic stacking tendency of purine bases.     

The initial step of DNA hairpin folding: a kinetic analysis using fluorescence correlation spectroscopy

Conformational fluctuations of single-stranded DNA (ssDNA) oligonucleotides were studied in aqueous solution by monitoring contact-induced fluorescence quenching of the oxazine fluorophore MR121 by intrinsic guanosine residues (dG). We applied fluorescence correlation spectroscopy as well as steady-state and time-resolved fluorescence spectroscopy to analyze kinetics of DNA hairpin folding. We first characterized the reporter system by investigating bimolecular quenching interactions between MR121 and guanosine monophosphate in aqueous solution estimating rate constants, efficiency and stability for formation of quenched complexes. We then studied the kinetics of complex formation between MR121 and dG residues site-specifically incorporated in DNA hairpins. To uncover the initial steps of DNA hairpin folding we investigated complex formation in ssDNA carrying one or two complementary base pairs (dC–dG pairs) that could hybridize to form a short stem. Our data show that incorporation of a single dC–dG pair leads to non-exponential decays for opening and closing kinetics and reduces rate constants by one to two orders of magnitude. We found positive activation enthalpies independent of the number of dC–dG pairs. These results imply that the rate limiting step of DNA hairpin folding is not determined by loop dynamics, or by mismatches in the stem, but rather by interactions between stem and loop nucleotides.     

Hairpin and duplex formation in DNA fragments CCAATTTTGG, CCAATTTTTTGG, and CCATTTTTGG: a proton NMR study

Three DNA fragments, CCAATTTTGG (1), CCAATTTTTTGG (2), and CCATTTTTGG (3), were studied by proton NMR spectroscopy in aqueous solution. All these oligodeoxyribonucleotides contain common sequences at the 5' and 3' ends (5'-CCA and TGG-3'). 2 as well as 3 forms only hairpin structures with four unpaired thymidylyl units, four and three base pair stems, respectively, in neutral solution under low and high NaCl concentrations. At high salt concentration the oligomer 1 forms a duplex structure with -TT- internal loop. On the other hand, the same oligomer forms a stable hairpin structure at low salt and low strand concentrations at pH 7. The hairpin structure of 1 has a stem containing only three base pairs (CCA.TGG) and a loop containing four nucleotides (-ATTT-) that includes a dissociated A.T base pair. The two secondary structures of 1 coexist in an aqueous solution containing 0.1 M NaCl, at pH 7. The equilibrium shifts to the hairpin side when the temperature is raised. The stabilities and base-stacking modes of all three oligonucleotides in two different structures are reported.     

The effect of hydrostatic pressure on the thermal stability of DNA hairpins

DNA hairpins consist of two distinct structural domains: a double stranded stem and a single-stranded loop that connect the two strands of the stem. Previous studies of short DNA hairpins have revealed that loop and stem sequences can significantly affect the thermodynamic stability of short DNA hairpins. In this work we present the effect of hydrostatic pressure on the helix-coil transition temperature (T_M) for 11 16-base, hairpin-forming DNA oligonucleotides. All of the samples form a hairpin with a 6-base pair stem and a four-base loop. In addition, the four base pairs at the end of the stem distal from the loop are the same for every molecule. We have varied loop sequence and identity of the two duplex base pairs adjacent to the loop. Using the change in UV absorption to monitor the conformational state of the oligonucleotide the hairpin-coil transition temperature of these molecules was studied as a function of sodium ion concentration and pressure. From these data we calculated the volume change accompanying the transition. Model-dependent (van't Hoff) transition parameters such as ΔH_vH and transition volume (ΔV) were estimated from the analysis of conformational transitions. Experiments revealed that the ΔV for denaturation of these molecules range from − 2.35 to + 6.74 cm³ mol⁻¹. The expansibility (ΔΔV/ΔT) and the pressure dependence of cation release are also presented. The difference in the volume change for this transition is related to the differences in the hydration of these molecules.