Radiation effects of poly(l-glutamic acid) and poly(l-lysine) in the helix-coil transitional state

Summary The degradation of poly(l-glutamic acid) and poly(l-lysine) by X-rays and ultraviolet radiation has been studied at variouspH values, including the helix-coil transition region. It was found that X-rays cause maximum degradation in the transition region for both polypeptides, indicating that the efficiency of degradation is affected not only by the secondary structure but also by some other factors related to the transition. On the contrary, the degradation by monochromatic ultraviolet 2537 radiation showed a strong dependence on helix content of PLGA, suggesting that degradation occurs more effectively in the helical form than in the coil form. Theoretical calculations were made to approximate thepH dependence of degradation, assuming different cross sections for the peptide bonds in helix, coil, and junctions between helical and coil regions.     

A calorimetric study of the triple helix formation: interaction of poly(C) - guanosine - protonated poly(C).]

The triple helix formation of poly(C) - guanosine - poly(C+) was investigated by the help of an LKB scanning micro-calorimeter. The existence of the triple helix could also be shown by recording the melting curves. The ultraviolet absorption at different wave lengths namely 275 nm, 260 nm, and 245 nm was plotted as a function of the temperature. Furthermore formation of the triple helix was shown by plotting the ultraviolet absorption at 245 nm during the increasing addition of guanosine solution to a fixed amount of poly(C) in the solution. Finally the formation of the triple helix was demonstrated by plotting the ultraviolet absorption at 245 nm of a certain mixture of the components while the pH value of the solution was continuously lowered. All these methods show that the monomer interacts with the polymer double helix to form a triple helix. The calorimetric measurements show that the reaction enthalpy is concentration dependent. Above a threshold concentration a rapid increase of the reaction enthalpy is observed. This increase occurs in a very narrow concentration interval. Above this interval a final value of the reaction enthalpy is reached. The amount of the reaction enthalpy for the interaction of guanosine with poly(C) - poly(C+) double helix is 5.5 Kcal (mol base triplet)-1.     

Spectroscopic and equilibrium dialysis studies of poly(α-L-glutamic acid)–Cu(II) complexes in the pH range 4–7

The formation of complex between the Cu²⁺ ion and poly(α-L -glutamic acid) [poly(Glu)] in 150 mM NaCl solutions was studied by uv–visible absorption and equilibrium dialysis methods at the mixing ratios of Glu residues to Cu²⁺, R, of 32, 16, and 8 and in the pH range 4–7. The results showed that more than 90% of Cu²⁺ ions bind to the poly(Glu) at pH > 4.9, but the bound Cu(II) begins to dissociate with a decrease in pH. The absorption spectra of bound Cu(II) varied with pH and R in a complicated manner. Three different component spectra were disclosed from the analysis of the pH dependence of the bound spectra. We concluded that poly(Glu)–Cu(II) complexes fall into three classes in the pH range 4–7, with the proportions of these complexes varying with both pH and R. The three complexes predominate either in the helix or extended-coil region, in the helix–coil transition region, or in the helix-aggregate region. The stability constant and binding mode of each Cu(II)–Glu complex were estimated from the dialysis data. With these results, the possible structure of each complex is discussed.     

Comparison of the helix–coil conformational changes of poly(deoxyadenylate-deoxytymidylate) and poly(deoxyadenylate-deoxythymidylate) induced by variations of hydrogen-ion concentration

Double-helical poly(dG-dC) and poly(dA-dT) are DNA analogs in which the interactions between the two strands of the helix are, respectively, either the stronger G/C type or the weaker A/T type along the entire length of macromolecules. Thus, these synthetic polynucleotides can be considered as representatives of the most stable and the least stable DNA. In the investigations presented here, potentiometric titrations and stopped-flow kinetic experiments were carried out in order to compare the pH-induced helix–coil conformations (10°C and 150mM [Na⁺]) the pH of the helix–coil transition (pH_m) is 12.81 for poly(dG-dC) and 11.76 for poly(dA-dT). The unwinding of double-helical poly(dG-dC) initiated by a sudden change in pH was found to be a simple exponential process with rate constants in the range of 200–600 sec^?1, depending on the final value of the pH jump. The intramolecular double-helix formation of poly(dG-dC) was studied by lowering the pH of the solutions from a value above pH_m to that below pH_m in dilute solutions (15.5 ug/ml [polymer]). Under these conditions, the observed rewinding reactions displayed a major and two exponential phases, all of which were independent of polymer concentration. From the comparison of the results of poly(dA-dT) and poly(dG-dT) would unwind faster than poly(dG-dC). However, if the pH jumps are such that they present the same perturbation of these polymers relative to their pH_m values, no significant differences exist between the rates of helix–coil conformation changes of poly(dA-dT) and poly(dG-dC).     

Far-infrared absorption of nucleotides and poly(I)·poly(C) RNA

We have measured the ir absorption of 5′CMP, 5′IMP, and poly(I)·poly(C) from ～25 to ～500 cm^?1. From a comparison of the data with the previously measured absorption of the corresponding nucleosides and bases we can identify several “lines” associated with the deformation of the ribose ring. Out-of-plane deformation of the bases contributes strongly to vibrations near 200 cm^?1. The same ribose vibrations observed in the nucleotides are found in poly(I)·poly(C). They sharpen with increasing water absorption. A study of the spectra of poly(I)·poly(C) as a function of the adsorbed water indicates that water does not contribute in a purely additive fashion to the polynucleotide spectrum but depends on the conformation of the helix. However, the only spectral feature that shifts drastically with conformation is near 45 cm^?1. Measurements at cryogenic temperatures indicate some sharpening of the spectrum of poly(I)·poly(C). Instead, no sharpening is observed in the spectrum of the nucleotides. Shear degradation of poly(I)·poly(C) produces significant spectral changes in the 200-cm^?1 region and sharpening of the features assigned to the low-frequency ribose-ring vibrations.     

Conformational changes induced in ionized poly(L-arginine) and poly(L-histidine) by sodium dodecyl sulfate

Circular dichroism spectra have been obtained for cationic poly(L -arginine) and poly(L -histidine) in aqueous solutions containing varying amounts of sodium dodecyl sulfate. The detergent induces a disorder-order transition in both polypeptides. In each case the transition is cooperative and occurs when the ratio of detergent to amino acid residue is near unity. The ordered structure formed by poly(L -arginine) is readily identifiable as an α helix. Poly(L -histidine) appears to form a β structure in which the 211-nm electronic absorption band of the imidazole group exhibits significant rotatory strength.     

Synthesis and conformational study of poly(N delta-carbobenzoxy, N delta-benzyl-L-ornithine) and poly(N delta-benzyl-L-ornithine)

Poly(N^δ-carbobenzoxy, N^δ-benzyl-L -ornithine) (PCBLO) was prepared by the standard NCA method. PCBLO was converted into poly(N^δ-benzyl-L -ornithine) (PBLO) through decarbobenzoxylation with hydrogen bromide. The monomer N^δ-benzyl-L -ornithine was synthesized by reacting L -ornithine with benzaldehyde, followed by hydrogenation. The conformation of the two polypeptides was studied by optical rotatory dispersion and circular dichroism. PCBLO forms a right-handed helix in helix-promoting solvents. In mixed solvents of chloroform and dichloroacetic acid (DCA) it undergoes a sharp helix–coil transition at 12% (v/v) DCA at 25°C, as compared with 36% for poly(N^δ-carbobenzoxy-L -ornithine) (PCLO). Like PCLO, the helix–coil transition is “inverse,” that is, high temperature favors the helical form. PBLO is soluble in water at pH below 7 and has a “coiled” conformation. In 88% (v/v) 1-propanol above pH (apparent) 9.6 it is completely helical. In 50% 1-propanol the transition pH (apparent) is about 7.4; this compares with a pH_tr of about 10 for poly-L -ornithine in the same solvent.     

Circular dichroism of collagen, gelatin, and poly(proline) II in the vacuum ultraviolet.

Circular dichroism spectra for acid-soluble calfskin collagen, gelatin, and poly(proline) II in solution have been extended into the vacuum ultraviolet region. The extended spectrum of gelatin reveals that the circular dichroism of this unordered polymer is more closely related to the spectrum of charged polypeptides than might be evident from near ultraviolet work. A short-wavelength band is found at about 172 nm, which corresponds in position, magnitude, and sign to a band recorded earlier for poly(L -glutamic acid) at pH 8.0. This band is observed in a helical structure for the first time in the vacuum ultraviolet circular dichroism and absorption spectra of poly(proline) II. Both circular dichroism and absorption spectra point to the assignement of this band as the nσ*. Neither the nσ* nor the expected positive lobe of the ππ* helix band is observed in the extended circular dichroism spectrum of collagen. We postulate that these two bands cancel here in analogy to the case of α-helical poly(L -glutamic acid).     

13C Nuclear magnetic resonance study of salt induced conformational change of basic polypeptides: Poly (L-lysine), poly (L-arginine), and poly (L-ornithine)

A ¹³C-nmr study of the salt-induced helix–coil transition of the basic polypeptides poly(L -lysine) [(Lys)_n], poly(L -arginine) [(Arg)_n], and poly (L -ornithine) [(Orn)_n] was performed to serve as a reference of the helical portion of histones and other proteins. As is the case with pH-induced helix–coil transition, the downfield displacement of the C^α and carbonyl carbon signals are observed in the helical state. The upfield shift of the C^β signals, on the other hand, is noted in the salt-induced transition. Regardless of the differences in the side chains and also the salts used, very similar helix-induced chemical shifts are obtained for (Lys)_n and (Arg)_n. However, the displacement of the C^α, C^β, and carbonyl carbons of (Orn)n in the presence of 4M NaClO₄ is found to be almost 50% of that of (Lys)_n and (Arg)_n. This is explained by the fact that the maximum helical content is about 50%, consistent with the ORD result. Further, the motion of the backbone and side chains of the helical from was estimated by measuring the spin-lattice relaxation time (T₁), nuclear Overhauser enhancement (NOE), and line width. In the case of (Lys)_n, the motion of the side chains is charged very little in comparison with that of the random coil. Indicating that the aggregation of the salt-induced helix is small in contrast to that of the pH-induced helix. For (Arg)_n, however, the precipitate of the helical polymers is mainly due to aggregation.     

Lattice vibrational modes of poly(rU)-poly(rA). A coupled single-helical approach

The eigenvalues and eigenvectors of 11-fold double-helical poly(rU)·poly(rA) have been calculated. The vibrational potential energy of the double-helical structure is initially considered to be a sum of the vibrational potential energy of the single-helical structures poly(rU) and poly(rA). Coupling between the single helices is introduced by including a stretch force constant for each hydrogen bond between the uracil and adenine base residues. In addition, a model is presented for nonbonded interactions between nearest neighbor base pairs, which is consistent with a previous model for such interactions in the single helices. Because of the simple structural relationship between the uncoupled single helices and the double helix we are able to cast the secular equation for poly(rU)·poly(rA) in a form suitable for the use of perturbation theory using the previously calculated normal modes for the single helices as the unperturbed modes. Perturbation theory was found to be inapplicable for the region of the spectrum ?450 cm^?1. In this region an exact Green function technique is used to calculate the strongly coupled modes. We explicitly display one aspect of these double-helical normal modes. The stretching motions of the hydrogen bonds in the region of the spectrum <450 cm^?1 have been plotted as bar graphs for each mode.     

Microwave absorption of DNA between 8 and 12 GHz

The microwave absorption of aqueous solutions of DNA extracted from E. coli has been studied between 8 and 12 GHz by the use of an optical heterodyne technique. By measuring optically the temperature rise produced in an absorbing sample by pulsed microwave radiation, unambiguous, direct measurement of the microwave absorption is possible. Our results show that E. coli DNA absorbs microwaves in the 8–12-GHz region substantially more efficiently than water, which is itself an extremely efficient absorber. The observed absorption is featureless and decreases slightly with increasing frequency. These observations are consistent with an explanation involving direct absorption by longitudinal acoustic modes of the double helix.     

Kinetic studies of the helix–coil transition in aqueous solutions of poly(L-lysine)

The rate of conformational change of aqueous poly(α-L -lysine) solutions was measured using the electric field pulse relaxation method with conductivity detection. The relaxation time as a function of pH exhibits two maxima. One is assigned to a proton transfer reaction and the other to the helix–coil conformational transition. The helix nucleation parameter and the maximum relaxation time yield the rate constant of helix growth process (k_F) according to Schwarz's kinetic theory as k_F = 2 × 10⁷ sec^?1, which is comparable to that of the poly(glutamic acid) solution. The thermodynamic parameters of the helix growth process are compared with those of poly(glutamic acid).     

Interaction of poly(X) with poly(U): alkali metal ion specificity suggests a four-stranded helix

Interaction of poly(X) with poly(U) to form a regular, ordered structure has been investigated by uv, CD, and ir spectroscopy. The XU complex exhibits a marked dependence of T_m on the nature of the alkali metal ion present in solution. Stability of the complex is surprisingly high and increases with ionization of X residues at N3. The ir evidence shows the U residues are present as the diketo form and X residues as the 6-keto-2-enolate anion and indicates that the X and U carbonyl groups are vibrationally coupled. A mixing curve exhibits a single minimum, at 1:1, but we conclude that the actual combining ratio is twice this value and that the formula of the complex is X₂U₂. We propose a model based on a four-standard helix with size-specific alkali metal complexing to X and U carbonyl oxygens in the axial channel of the helix. This model and previously suggested two-stranded structures are evaluated in terms of the physical and chemical properties of the complex.     

The structure of triple helical poly(U).poly(A).poly(U) studied by Raman spectroscopy

Using Raman spectroscopy, we examined the ribose-phosphate backbone conformation, the hydrogen bonding interactions, and the stacking of the bases of the poly(U).poly(A).poly(U) triple helix. We compared the Raman spectra of poly(U).poly(A).poly(U) in H2O and D2O with those obtained for single-stranded poly(A) and poly(U) and for double-stranded poly(A).poly(U). The presence of a Raman band at 863 cm-1 indicated that the backbone conformations of the two poly(U) chains are different in the triple helix. The sugar conformation of the poly(U) chain held to the poly(A) by Watson-Crick base pairing is C3' endo; that of the second poly(U) chain may be C2' endo. Raman hypochromism of the bands associated with base vibrations demonstrated that uracil residues stack to the same extent in double helical poly(A).poly(U) and in the triple-stranded structure. An increase in the Raman hypochromism of the bands associated with adenine bases indicated that the stacking of adenine residues is greater in the triple helix than in the double helical form. Our data further suggest that the environment of the carbonyls of the uracil residues is different for the different strands.     

Helix-helix transitions in DNA: fibre X-ray study of the particular cases poly(dG-dC) · poly(dG-dC) and poly(dA) · 2poly(dT)

The helix-helix transitions which occur in poly(dG-dC) · poly(dG-dC) and in poly (dG-m⁵dC) · poly(dG-m⁵dC) are commonly assumed to be changes between the right-handed A- or B-DNA double helices and the left-handed Z-DNA structure. The mechanisms for such transconformations are highly improbable, especially when they are supposed to be active in long polynucleotide chains organised in semicrystalline fibres. The present alternative possibility assumes that rather than the Z-DNA it is a right-handed double helix (S-DNA) which actually takes part in these form transitions. Two molecular models of this S form, in good agreement with X-ray measurements, are proposed. They present alternating C(2′)-endo and C(3′)-endo sugar puckering like the “alternating B-DNA” put forward some years ago. Dihedral angles, sets of atomic coordinates and stereo views of the two S-DNA structures are given, together with curves of calculated diffracted intensities. Furthermore, we question the possibility of obtaining semicrystalline fibres with triple helices of poly(dA) · 2poly(dT) in a way which renders X-ray diffraction efficient. It is suggested that, up to now, only double helices of poly(dA) · poly(dT) can actually be observed by fibre X-ray diffraction measurements. Received: 30 March 1999 / Revised version: 30 June 1999 / Accepted: 30 June 1999     

The thermal induced helix--beta transition of poly(N epsilon-methyl-L-lysine) and poly(N delta-ethyl-L-ornithine) in aqueous solution

Uncharged poly(N^ε-methyl-L -lysine) (PMLL) and its isomer, poly(N^δ-ethyl-L -ornithine) (PELO), in alkaline solution (pH ca. 12) undergo a helix-to-β transition upon mild heating at 50°C or higher in a manner similar to that of poly(L -lysine) (PLL). The rate of conversion follows the order: PMLL < PELO < PLL. The helix can be regenerated upon cooling near zero degrees, for instance, after more than 12 hr at 2°C. At concentrations less than 0.02% the β form is intramolecular, but at higher concentrations both intra- and intermolecular β forms are generated. Poly(N^δ-methyl-L -ornithine) (PMLO), an isomer of PLL, behaves like poly(L -ornithine); uncharged PMLO in alkaline solution is partially helical and becomes disordered at elevated temperatures.     

Base tilt of poly[d(A)]-poly[d(T)] and poly[d(AT)]-poly[d(AT)] in solution determined by linear dichroism

We have measured the CD, isotropic absorption, and LD of poly[d(A)]–poly[d(T)] and poly[d(AT)]–poly[d(AT)] in the vacuum-uv spectral region. The reduced dichroism (LD divided by isotropic absorption) varied as a function of wavelength and was independent of shear gradient. Thus, the bases are not perpendicular to the helix axis in solution. Since the directions of the transition dipoles are known, the orientations of the bases in the polymers can be calculated from the reduced dichroism spectra. The results show that the base normals are tilted at angles greater than 25°, with respect to the helix axis, and thymine is tilted more than adenine for both polymers. The tilt axes of adenine and thymine are not parallel, indicating a large propeller twist. Space-filling models of poly[d(A)]–poly[d(T)] and poly[(AT)]–poly[d(AT)] are built based on our results, and the conformations of the two (A + T) polymers in solution are discussed.     

Dependence on ionic strength of charge-induced coil-helix transition of sodium poly(L-glutamate) in aqueous solution

Helix contents of sodium poly(L-glutamate) in aqueous NaCl solutions were estimated as functions of the degree of ionizalion, alpha. and the salt concentration by CD measurement. The helix content increases with increasing salt concentration but this helix stabilization decreases with decreasing alpha and at alpha<0.25 the helix conformation is destabilized by salt addition. The alpha dependence of the helix stabilization was qualitatively interpreted by Manning's theory in which electrostatic interactions between charges on alternatively arrayed coil and helix segments in a simplified polymer model were incorporated.     

Interaction of sodium decyl sulfate with poly(L-ornithine) and poly(L-lysine) in aqueous solution

The binding isotherms of sodium decyl sulfate to poly(L -ornithine), poly(D ,L -ornithine), and poly(L -lysine) at neutral pH were determined potentiometrically. The nature of a highly cooperative binding in all three cases suggests a micelle-like clustering of the surfactant ions onto the polypeptide side groups. The hydrophobic interaction between the nonpolar groups overshadows the coulombic interaction between the charged groups. The titration curves can be interpreted well by the Zimm–Bragg theory. The average cluster size of bound surfactant ions is sufficiently large to promote the β-structure of (L -Lys)_n even at a very low binding ratio of surfactant to polypeptide residue, whereas the onset of the helical structure for (L -Orn)_n begins after about 7 surfactant ions are bound to two turns of the helix. The CD results are consistent with this explanation.     

Raman spectroscopic measurements in self‐pressurized aqueous solutions above 100°C: The melting of poly(G) and poly(G) · poly(C)

We have studied by Raman spectroscopy the thermal behavior of associated polyguanylic acid [poly(G)] and polyguanylic–polycytidylic acid [poly(G) · poly(C)] in self‐pressurized aqueous solutions contained in sealed capillary tubes. The associated polynucleotides were found to be very resistant to heat, but evidence of thermal degradation was observed after melting of the helical structures. The cooperative melting transition of the four‐stranded complex of poly(G) was located at 141°C in 0.5M KCl, 135°C in 0.5M NaCl, 129°C in 0.5M LiCl, 123°C in 0.1M tetramethylammonium perchlorate, and 105°C in 0.1M tetraethylammonium bromide solutions. The transition was observed at 130°C in poly(G) · poly(C) (in 0.5M NaCl). The results in this case show that a four‐stranded poly(G) complex is formed following the melting of the double helix. © 1999 John Wiley & Sons, Inc. Biopoly 49: 21–28, 1999