Experimental thermodynamics of the helix–random coil transition. II. Calorimetric measurement of transition enthalpies of poly A in acid aqueous solution

The enthalpy change accompanying the double helix–coil transition of polyriboadenylic acid (poly A) in aqueous solution has been measured optically and calorimetrically in the pH range 5.7–4.5. The course of this cooperative transition was followed optically by measuring changes in ultraviolet absorption as a function of temperature at different pH values, and calorimetrically by determining the heat capacity of the solution through the transition region. From the latter measurements, the enthalpy of transition was calculated. It is shown, that ΔH is dependent on pH as it is expected from the influence of protonation of the double helix of poly A.     

Helix–coil transition of poly(α,L-glutamic acid) at an interface: Correlation with static and dynamic membrane properties

When adsorbed from an aqueous dilute solution at high pH into the pores of an inert cellulose acetate filter, poly(α,L -glutamic acid) remains strongly anchored to the pore walls. The existence of the helix–coil transition for the adsorbed polypeptide in a certain pH range is evidenced by static and dynamic membrane properties displayed by the “activated” filter, such as excess cation uptake, membrane potential, and hyraulic permeability. In particular, the variations of the hydrodynamic thickeness present a sigmoidal shape characteristic of the helix–coil transition at the interface, a transition apparently less sharp than in solution.     

Interaction of poly- ,L-glutamic acid with acridine orange

Interaction of poly-α,L -glutamic acid (PGLA) with acridine orange (AO) was studied with circular dichroism and absorption spectra measurements. The following results were observed: (1) the addition of a comparable amount of AO with the glutamyl residue to the PLGA solution at pH of 4.5 reduced the fraction of helix of the polymer; (2) when AO was added to the PGLA solution, the pH range of the helix-coil transition of the polymer shifted toward higher pH regions; and (3) when the mixture of the same amount of AO and the glutamyl residue was brought to the neutral and alkaline pH region, some induced circular dichroism bands were observed. In this case, it was assumed that PLGA in the system takes a helical form due to the neutralization of the anionized side chains by the cationic species of AO. We concluded that AO molecules bound to the carboxylate groups of the side chains of PLGA arrange to from a righthanded super-helix which surrounds the core of the righthanded α-helix of PLGA.     

Mechanical properties of poly(glutamic acid) in aqueous solution

The interaction between ionizable carboxyl groups and the conformation of poly-(glutamic acid) (PGA) in aqueous solution were investigated by the mechanical method. The dynamic rigidity of the PGA solution has a maximum value at the pH corresponding to about 50% neutralization point. This may be due to establishing of a maximum attractive force by proton/charge fluctuation between ionizable carboxyl groups at that pH. The dynamic viscosity has a sharp change in the region of pH 5.5–6.5. It is suggested that this behavior is due to the helix–coil transition.     

Total optical activity of agarose: relation to observable transitions in the vacuum ultraviolet

The changes in optical activity that accompany and characterize the coil-helix and helix-coil transitions of agarose in aqueous solutions and gels have been investigated by combined quantitative analysis of data from vacuum ultraviolet circular dichroism (VUCD) and optical rotary dispersion (ORD). VUCD of agarose in the high-temperature coil state shows a single accessible Gaussian band centered at ～183 nm. In the helix state this band is blue-shifted by ～9 nm, and the intensity is increased by a factor of ～2.6. Spectra at intermediate temperatures can be fitted to within experimental error by linear combination of coil and helix spectra, the relative proportions required providing an index of the extent of conformational ordering. ORD spectra throughout the conformational transition have a common form and differ only in absolute magnitude. The temperature course of conformational ordering derived from ORD intensity is in close agreement with the values obtained from VUCD. In both the coil and helix states the accessible VUCD band is positive, while the overall ORD is negative, indicating strong negative CD activity at lower wavelength. The ORD contribution corresponding to the positive VUCD band was calculated by Kronig–Kramers transform, and it was subtracted from the total ORD to give the residual ORD from all other optically active transitions of the molecule. In both the coil and helix states, this residual ORD could be fitted to within experimental error by a single Gaussian CD band at ～149 nm. A negative band at this wavelength has been reported previously for agarose films, but the observed intensity, relative to that of the lower energy positive band, is substantially smaller than the fitted value under hydrated conditions. In both the coil and helix states the total optical activity of agarose, characterized by observed ORD spectra, can be matched to within experimental error by Kronig-Kramers transform of the 149-nm negative band and the smaller positive band at higher wavelength, with no necessary involvement of deeper-lying transitions. The significance of this conclusion for fundamental understanding of carbohydrate optical activity is discussed.     

Studies of the helix–coil transition of poly-L-lysine in film and solution

Water-insoluble films of poly-L -lysine, crosslinked with formaldehyde, were suspended in aqueous media and their relative lengths measured as a function of pH. A sharp transition of the polymer was observed in the pH range which corresponded with that observed in polylysine solutions by optical rotation or dilatometry. In NaBr and NaCl solutions the coiled form of the polylysine film shrinks with increasing salt concentration, but in NaHCO₃ solution the extent of the contraction is larger, and the coil–helix transition of polylysine occurs at lower pH when NaHCO₃ is added to the medium. If one assumes the formation of amino carbamate in this case, this phenomenon can be well explained. Urea does break up the hydrogen bonds in helical polylysine film, but not completely. This result is interesting compared with that obtained for poly(L -glutamic acid). After the coil–helix transition region was found by film experiments, the volume change associated with the coil-to-helix transition was measured and found to be about 1–l.5 ml. per amino residue after taking electrostatic interaction into consideration. This value is nearly same as that obtained for poly(L -glutamic acid). By contrast, the value for poly-γ-benzyl-L -glutamate was reported to be ?0.077 ml./mole of repeating unit. So it is still necessary to determine the magnitude and direction of the volume change for various kinds of polypeptides.     

High-resolution study of the helix–coil transition of poly(L-glutamic acid): Spectroscopic data correlation and the discovery of a new transition

This article reports on both (1) the precision and capability of a computerized multidimensional spectrophotometric system recently developed in our laboratory and (2) the high-resolution study of the helix–coil transition of poly(L-glutamic acid)[poly(Glu)], especially with regard to the discovery of an overlooked transition which is attributable to order–disorder rearrangement of the poly(Glu) side chain in the α-helical conformation. This study was made possible by the high performance of the system used. The simultaneous and continuous measurement of the circular dichroism, the absorbance and light-scattering intensity, and the pH titration curve of poly(Glu) in aqueous salt solution was carried out under continuous scanning of pH ranging from 8 to 2. Besides the well-known random coil to α-helix transition that occurs at about pH 5.5, a highly cooperative transition, which is indicated as a small but definite step in several spectral dimensions, is observed for the first time at pH 4.3. The transition is ascribed to an order–disorder conversion of the side chain on the α-helix backbone.     

Electric birefringence studies on polyglutamic acid.

The electric birefringence for the aqueous solution of poly-L -glutamic acid (PGA) in the helical form was studied. PGA samples were fractionated by gel column chromatography. PGA showed a positive electric birefringence. The permanent dipole moment of the PGA molecule was suggested to be largely suppressed. The measurements of the intrinsic Kerr constants for various molecular lengths showed that the electric anisotropy (polarizability) of PGA is proportional to the 1.5 power of the length. The electric birefrigence measurement was also carried out in the helix–coil transition region. The Kerr constant of PGA was largely reduced on going from the helical form to the coiled form.     

Stable and metastable forms of poly(G)

Poly(G) is shown by ir spectroscopy to be capable of existence in a metastable form which is converted spontaneously at ambient temperature, or more rapidly on heating, to a stable form. The metastable form can be regenerated by freezing and thawing the solution. The high-charge density of four-stranded poly(G) makes it especially susceptible to electrostatic destabilization by use of Et₄N⁺ counterions, which screen electrostatic repulsion of multiple strands less effectively than alkali metal ions. Poly(G) has been obtained for the first time in the single-stranded form in aqueous solution and shown to undergo a fully reversible helix–coil transition on heating.     

pH- and thermal-dependent conformational transition of PGAIPG, a repeated hexapeptide sequence from tropoelastin

The secondary structure of PGAIPG (Pro-Gly-Ala-IIe-Pro-Gly), a repeated hexapeptide of tropoelastin, in buffer solution of different pH was determined by using attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. The thermal-dependent structural change of PGAIPG in aqueous solution or in solid state was also examined by thermal FTIR microspectroscopy. The conformation of PGAIPG in aqueous solution exhibited a pH-dependent structural characterization. A predominant peak at 1614 cm(-1) (aggregated beta-sheet) with a shoulder near 1560 cm(-1) (beta-sheet) appeared in pH 5.5-8.5 buffer solutions. A new broad shoulder at 1651 cm(-1) (random coil and/or alpha-helix) with 1614 cm(-1) was observed in the pH 4.5 buffer solution. However, the broad shoulder at 1651 cm(-1) was converted to a maximum peak at 1679 cm(-1) (beta-turn/antiparallel beta-sheet) when the pH shifted from 4.5 to 3.5, but the original pronounced peak at 1614 cm(-1) became a shoulder. Once the pH was lowered to 2.5, the IR spectrum of PGAIPG was dominated by major absorption at 1679 cm(-1) with a minor peak at 1552 cm(-1) (alpha-helix/random coil). The result indicates that the pH was a predominant factor to transform PGAIPG structure from aggregated beta-sheet (pH 8.5) to beta-turn/intermolecular antiparallel beta-sheet (pH 2.5). Moreover, a partial conformation of PGAIPG with minor alpha-helix/random coil structures was also explored in the lower pH buffer solution. There was no thermal-dependent structural change for solid-state PGAIPG. The thermal-induced formation of aggregated beta-sheet for PGAIPG in aqueous solution was found from 28 to 30 degrees C, however, which might be correlated with the formation of an opaque gel that turned from clear solution. The formation of aggregated beta-sheet structure for PGAIPG beyond 30 degrees C might be due to the intermolecular hydrogen bonded interaction between the hydrophobic PGAIPG fragments induced by coacervation.     

The effects of ultra-violet light and X-rays on aqueous solutions of poly-L-glutamic acid

Summary Poly-L-glutamic acid (PLGA) in aqueous solution in helical or random coil form was irradiated in air by X-rays or ultra-violet light. It was observed that both X-rays and ultra-violet light caused degradation of PLGA in either form. The changes in molecular weight of PLGA in alkaline solutions caused by X-ray irradiation were larger than those in acidic solutions. This fact indicates that the coil form suffers more degradation than the helix form. X-rays caused little change in the conformation of PLGA, while ultra-violet light effectively broke the helix form. The decrease in helix content brought about by ultra-violet exposure could not be explained just by degradation.     

Proton nuclear magnetic resonance investigations of the nucleation and propagation reactions associated with the helix-coil transition of d-ApTpGpCpApT in H2O solution.

The chemical shifts and line widths of the Watson-Crick ring NH resonances of the self-complementary duplex of d-ApTpGpCpApT have been monitored in the presence of 0.1 M phosphate at neutral pH in aqueous solution. While the resonance positions of the terminal and internal AT base pairs shift upfield and broaden as average resonances with increasing temperature (helix and coil exchange several times prior to exchange with water from the coil form), those of the central GC base pairs broaden in the absence of upfield shifts (exchange with water occurs each time helix converts to coil). The line-width changes at the AT base pairs monitor the lifetime of the coil state at these positions prior to exchange with water while the line-width changes at the GC base pairs monitor the lifetime of the helix prior to dissociation to strands. This permits the separation of the propagation reaction at the AT base pairs from the nucleation reaction at the GC base pairs during helix formation. The experimental data have been quantitatively analyzed to yield (at 20 degrees) a nucleation formation rate of approximately 10(3) sec-1 for the GC base pairs (bimolecular rate constant of approximately 6 times 10(6) l. mol-1 sec-1) and a dissociation rate of 6 times 10(2) sec-1 at these same base pairs (unimolecular dissociation to strands). The unimolecular propagation reactions at the terminal and terminal base pairs are associated with reaction rates greater than 10(4) sec-1. These values are consistent with a slow formation of a stable nucleus at the GC base pairs followed by a rapid propagation reaction at the AT base pairs. The line width of the (GC) central base pairs in the presence of phosphate is a direct measure of the lifetime of the total helix and yields an activation energy of 45 kcal for helix to coil conversion measured over a narrow temperature range. The exchange from the coil form with water is catalyzed by 0.1 M phosphate with a rate constant kHPO2-/4 = 0.2 times 10(6) 1. mol-1 sec-1.     

Study of helix-coil transition of DNA by dielectric constant measurement

The thermal helix–coil transition of DNA was studied by means of dielectric constant measurements. The dielectric dispersion of native helical DNA is characterized by a large dielectric increment and a large relaxation time, whereas that of denatured coil DNA is characterized by a small dielectric increment and a small relaxation time. The dielectric dispersion of partially denatured DNA is of particular interest. At the intermediate stage of the helix–coil transition, dispersion curves which are different from either that of helix DNA or that of coil DNA appear. This is particularly pronounced for large DNA. This indicates the presence of an intermediate form of DNA. Flow birefringence measurements were carried out simultaneously. The negative birefringence of helical DNA diminishes as the helix–coil transition proceeds. However, the extinction angle remains constant, as long as it can be measured. These results indicate the absence of intermediate forms during the helix–coil transition. The discrepancy between dielectric and birefringence measurements can be resolved by assuming that the intermediate forms are not birefringent. The size distribution of native DNA and of the indicated intermediate form of DNA was studied. It is found that a logarithmic normal distribution function explains the distribution of size of DNA reasonably well.     

Comparison of helix stabilities of poly-L-lysine, poly-L-ornithine, and poly (L-diaminobutyric acid)

The helix–coil transitions of aqueous solutions of poly-α-L -lysine (PLL), poly-α-L -ornithine (PLO), and poly(α,γ-L -diaminobutyric acid) (PLDBA) have been investigated as functions of pH at 25°C and of temperature at pH 11.75, where these polymers are uncharged; in the cases of the latter two polyamino acids, the transitions have also been studied as functions of apparent pH in methanol-water solution (50/50 by volume). The helix stability of the polypeptides is shown to be a direct function of the number of methylene groups on the side chain. From an analysis of potentiometric titration data, we find that the difference between the helix stability of PLL and that of PLO is due to a difference of about 1 e.u. in the ΔS° of the transition. Combining the “melting curves” obtained from optical rotatory dispersion studies with the potentiometric titration data permits evaluation of the initiation parameter Z (or 1/σ^½) of the statistical mechanical theories for these transitions. The value obtained for Z in the case of uncharged aqueous PLO is ca. 35.     

Influence of methanol on the rotational diffusion of poly(L-lysine) in the helix–coil transition

¹³C spin-lattice relaxation times of poly(L -lysine) have been obtained at 67.9 MHz in aqueous solution and in a mixed solvent (40% methanol/60% water). A concomitant determination of the conformation by CD permits the correlation of conformation and rotational diffusion of the polymer. The dependence on pH of the spin-lattice relaxation times of the ¹³C^α and the side-chain carbon resonances reflects the diffusional motion in the random-coil conformation, in the helix–coil transition, and in the conformation of the α-helix. In the mixed solvent the reorientational correlation time of the C^α-H^α vector increases from τ = 0.37 nsec (random coil) to τ = 12.0 nsec (α-helix). In aqueous solution the correlation time of this vector increases from τ = 0.33 nsec (random coil) to τ ? 11 nsec. The reorientation rates of the side-chain methylene groups in the two solvents are markedly different. The reorientation of all methylene groups is reduced in the mixed solvent.     

A nuclear magnetic resonance study of the helix–coil transition of poly(L-glutamic acid)

There have been many reports that the nuclear magnetic resonance (nmr) spectra of a large number of polypeptides exhibit peak doubling of the α-carbon and the α-carbon proton in the helix–coil transition region. One apparent exception to this generalization has been polypeptides with ionizable side chains, where the helix–coil transition is induced by changes in pH in aqueous solution. Because it is important to establish the proper theoretical reason for the peak doubling and its relation to the rate of conformational change of amino acid residues, we have reexamined the proton and carbon-13 nmr spectra, at high field, for two polydisperse samples of poly(L -glutamic acid). Doubling of the α-carbon proton resonance as well as those of the α- and β-carbon, and backbone carbonyl are observed for a low-molecular-weight sample (DP = 54), while a higher molecular weight sample (DP = 309), exhibits only single resonances. Thus, polydispersity by itself is not sufficient to observe peak doubling; low-molecular weight is also required.     

Potentiometric titration of poly-S-carboxymethyl-L-cysteine in aqueous NaCl solution

pH titration measurements of poly- S-carboxymethyl-L -cysteine were undertaken in the aqueous Nacl solution in relation to the β form–random coil transition. The titration curves show a marked molecular weight dependence because of the shortened chain length of materials. Comparison of the optical rotatory dispersion parameter a₀ with the titration curve reveals that the titration curve apparently reflects a β structure–random coil transition. The β form of this polymer is assumed to be an intramolecular β form, rather than a β structure stabilized by an intermolecular hydrogen bond, at least in the polymer concentration range considered here. The standard free energy change per amino acid residue for the transition from un-ionized random coil to un-ionized β form is estimated to be about ?750 cal/mole residue in the range of 0.005–0.2M NaCl concentration.     

Thermodynamic model of secondary structure for α-helical peptides and proteins

A thermodynamic model describing formation of α-helices by peptides and proteins in the absence of specific tertiary interactions has been developed. The model combines free energy terms defining α-helix stability in aqueous solution and terms describing immersion of every helix or fragment of coil into a micelle or a nonpolar droplet created by the rest of protein to calculate averaged or lowest energy partitioning of the peptide chain into helical and coil fragments. The α-helix energy in water was calculated with parameters derived from peptide substitution and protein engineering data and using estimates of nonpolar contact areas between side chains. The energy of nonspecific hydrophobic interactions was estimated considering each α-helix or fragment of coil as freely floating in the spherical micelle or droplet, and using water/cyclohexane (for micelles) or adjustable (for proteins) side-chain transfer energies. The model was verified for 96 and 36 peptides studied by ¹H-nmr spectroscopy in aqueous solution and in the presence of micelles, respectively ([set I] and [set 2]) and for 30 mostly α-helical globular proteins ([set 3]). For peptides, the experimental helix locations were identified from the published medium-range nuclear Overhauser effects detected by ¹H-nmr spectroscopy. For sets 1, 2, and 3, respectively, 93, 100, and 97% of helices were identified with average errors in calculation of helix boundaries of 1.3, 2.0, and 4.1 residues per helix and an average percentage of correctly calculated helix—coil states of 93, 89, and 81%, respectively. Analysis of adjustable parameters of the model (the entropy and enthalpy of the helix—coil transition, the transfer energy of the helix backbone, and parameters of the bound coil), determined by minimization of the average helix boundary deviation for each set of peptides or proteins, demonstrates that, unlike micelles, the interior of the effective protein droplet has solubility characteristics different from that for cyclohexane, does not bind fragments of coil, and lacks interfacial area. © 1997 John Wiley & Sons, Inc. Biopoly 42: 239–269, 1997     

Ultrasonic absorption studies of poly-benzyl-L-aspartate in mixed solvents, in relation to the helix-cell transition

Ultrasonic absorption measurements were carried out on solutions of polybenzyl-L -aspartate (PBLA) in chloroform–dichloroacetic acid (DCA) and in 1,2-dichloroethane (DCE)–DCA, in the range 3.9–155 MHZ . The helix–coil transition of PBLA produces an increase of absorption which is larger in CHCl₃–DCA than in DCE–DCA solutions. The influence of the solvent on the excess ultrasonic absorption suggests that solvation processes may be involved in these changes of absorption. The plots of the absorption vs. the volume fraction of DCA do not show any absorption maximum. This indicates that the ultrasonic absorption is not sensitive to the helix–coil equilibrium of PBLA in the frequency range investigated. A maximum value of 10⁹ S ^?1 has been obtained for the rate constant of growth of a helix region.     

Proton and phosphorus NMR studies of d-CpG(pCpG)n duplexes in solution. Helix-coil transition and complex formation with actinomycin-D.

The Watson–Crick imino and amino exchangeable protons, the nonexchangeable base and sugar protons, and the backbone phosphates for d-CpG(pCpG)_n, n = 1 and 2, have been monitored by high-resolution nmr spectroscopy in aqueous solution over the temperature range 0°–90°C. The temperature dependence of the chemical shifts of the tetramer and hexamer resonances is consistent with the formation of stable duplexes at low temperature in solution. Comparison of the spectral characteristics of the tetranucleotide with those of the hexanucleotide with temperature permits the differentiation and assignment of the cytosine proton resonances on base pairs located at the end of the helix from those in an interior position. There is fraying at the terminal base pairs in the tetranucleotide and hexanucleotide duplexes. The Watson–Crick ring imino protons exchange at a faster rate than the Watson–Crick side-chain amino protons, with exchange occurring by transient opening of the double helix. The structure of the d-CpG(pCpG)_n double helices has been probed by proton relaxation time measurements, sugar proton coupling constants, and the proton chemical shift changes associated with the helix–coil transition. The experimental data support a structural model in solution, which incorporates an anti conformation about the glycosyl bonds, C(3) exo sugar ring pucker, and base overlap geometries similar to the B-DNA helix. Rotational correlation times of 1.7 and 0.9 × 10^?9 sec have been computed for the hexanucleotide and tetranucleotide duplexes in 0.1 M salt, D₂O, pH 6.25 at 27°C. The well-resolved ³¹P resonances for the internucleotide phosphates of the tetramer and hexamer sequences at superconducting fields shift upfield by 0.2–0.5 ppm on helix formation. These shifts reflect a conformational change about the ω,ω′ phosphodiester bonds from gauche-gauche in the duplex structure to a distribution of gauche-trans states in the coil structure. Significant differences are observed in the transition width and midpoint of the chemical shift versus temperature profiles plotted in differentiated form for the various base and sugar proton and internucleotide phosphorous resonances monitoring the d-CpG(pCpG)_n helix–coil transition. The twofold symmetry of the d-CpGpCpG duplex is removed on complex formation with the antibiotic actinomycin-D. Two phosphorous resonances are shifted downfield by ～2.6 ppm and ～1.6 ppm on formation of the 1:2 Act-D:d-CpGpCpG complex in solution. Model studies on binding of the antibiotic to dinucleotides of varying sequence indicate that intercalation of the actinomycin-D occurs at the GpC site in the d-CpGpCpG duplex and that the magnitude of the downfield shifts reflects strain at the O-P-O backbone angles and hydrogen bonding between the phenoxazone and the phosphate oxygens. Actinomycin-D is known to bind to nucleic acids that exhibit a B-DNA conformation; this suggests that the d-CpG(pCpG)_n duplexes exhibit a B-DNA conformation in solution.