Spectroscopic characterization of poly(Glu-Ala)

Infrared linear dichroism and ultraviolet circular dichroism speetroscopy have been used to distinguish four conformational forms of the ionizable sequential polypeptide poly(Glu-Ala). Two of these conformations, the α helix and the β form, were observed for the unionized polypeptide in solution. The α helix appeared immediately upon neutralization of the side-chain carboxyl functions, whereas the β form was observed after the neutralized solution had been standing for several days. The β form was also observed for films cast from either high or low pH solutions. Ionization of the glutamyl residues resulted in a circular dichroism spectrum which has previously been observed for charged homopolymers and appears to result from an extended helical conformation. Further, heating either the α helical or the charged extended helix resulted in a transition to a disordered chain. These results are consistent with the results of conformational calculations presented elsewhere.     

Conformational transitions and hydration of poly d (A-T) · poly d (A-T) in fibers

Conformational transitions of poly d(A-T) · poly d (A-T) have been studied by fiber X-ray diffraction and measurement of fiber dimensions. Results obtained for the D-A-B and D-B transitions are presented and analyzed. For all these form transitions, cooperativity effects are observed for the variation of the rise per nucleotide versus the relative humidity. Detailed information about hydration of the polynucleotide during form transitions and the numbers of water molecules per nucleotide necessary to stabilize the different helical conformations are presented. Offprint requests to: S. Premilat     

High pressure fourier transform infrared spectroscopy of poly(dA)poly(dT), poly(dA) and poly(dT)

The effect of hydrostatic pressure upon the DNA duplex, poly(dA)poly(dT), and its component single strands, poly(dA) and poly(dT) has been studied by fourier-transform infrared spectroscopy (FT-IR). The spectral data indicate that at 28 degrees C and pressures up to 12 kbar (1200 MPa) all three polymers retain the B conformation. Pressure causes the band at 967 cm(-1), arising from water-deoxyribose interactions, to shift to higher frequencies, a result consistent with increased hydration at elevated pressures. A larger pressure-induced frequency shift in this band is observed in the single stranded polymers than in the double stranded molecule, suggesting that the effect of pressure on the hydration of single strands may be greater than upon a double stranded complex. A pressure-dependent hypochromicity in the bands attributed to base stacking indicates that pressure facilitates the base stacking in the three polymers, in agreement with previous assessments of the importance of stacking in the stabilization of DNA secondary structure at ambient and high pressures.     

Study of specific interactions of amino acid esters with the synthetic polynucleotides poly(A) x 2 poly(U), poly(A) x poly(U) and poly(A) by thermal denaturation

The interactions of amino acid esters with poly(A)x2poly(U) and poly(A)xpoly(U) have been investigated by means of thermal denaturation of these polynucleotides. The esters under consideration raised the melting point, revealing the preferable binding to helical polynucleotide structures. The melting point shifts demonstrate the following sequence of the stabilities of these complexes: Arg greater than Lys much greater than His greater than Met greater than Ser greater than Gly. The same stability order is observed when studying the polynucleotide renaturation in the presence of esters. This order coincides with that previously obtained for the nucleotide base--amino acid ester complexes excepting basic amino acid esters. The ester interactions with poly(A) and poly(U) also reveal the specificity of monomer--monomer interactions. Some dynamic contributions into the studied specificity are also discussed.     

Structure of a pleiomeric form of poly d(AT):poly d(AT)

A chemically simple polynucleotide duplex, poly d(AT):poly d(AT), has been trapped in a fibrous form with a complex helical secondary structure with a large (7.4 nm) axial repeat 24 nucleotides long. The motif which is repeated by the symmetry elements is a hexanucleotide in which two residues (both TpA) have the less common gauche minus conformation at C3'-O3' and consequently distinctive phosphate orientations. This reinforces earlier conclusions that PypPu nucleotides tend to have different shapes from PupPy nucleotides and that DNA surfaces may signal what base sequences lie beneath them. The morphological differences between this pleiomeric DNA polymer and closely-related, but more symmetrical allomorphs are just as great as those observed in short DNA fragments in crystals.     

Poly (d trp-L Leu), poly (D Phe-L Leu) and poly (D Val-L Val-D Val-L Ala) as model polymers for the elucidation of the gramicidin a conformation

From infrared observations it is concluded that the conformation of poly (D Trp-L leu), poly (D Leu-L Trp), poly (D Phe-L leu) and poly (D val-L Val-D Val-L Ala) wich are model polymers of Gramicidin A is the same as one species of the antibiotic. Diffraction investigations indicate that poly-(D Phe-L Leu) and poly (D Val-L Val-D Val-L Ala) have a helical conformation which is most probably the pi DL 8 helix.     

Fluorescence decay of ethidium bromide in the presence of the Z-conformation of poly(dG-dC) and of poly(dG-dC) modified by chlorodiethylenetriamine platinum(II) chloride

A time-resolved fluorescence study of ethidium bromide (EB) in the presence of poly(dG-dC) and of poly(dG-dC) modified by chlorodiethylenetriamine platinum(II) chloride is presented under solvent conditions in which these polymers adopt the Z-conformation (high ionic strength). It is shown that these polynucleotides can intercalate a very small quantity of EB. The binding parameters have been determined. The fluorescence lifetime of EB is slightly higher when bound to the Z-conformation (?25 ns) than when bound to the B-conformation (?23.7 ns). The nature of the salt has been checked. In the presence of 2.5M NaClO₄, no transition from the Z-conformation to another conformation is observed when EB is added. On the contrary, in the presence of 4.25M NaCl, EB induces a cooperative transition from the Z-conformation to a conformation characterized by a much higher affinity for EB intercalation. In the case of poly(dG-dC) this last conformation is identical to the one observed at low ionic strength (B-conformation), but in the case of the platinated polymer this conformation is slightly different, as judged by the smaller value of the fluorescence lifetime of the intercalated EB.     

Spectroscopic studies on the structure of poly(8-bromoadenylic acid): Effect of glycosidic torsion angle on the conformation and flexibility in polyribonucleotides

The helix–coil transition and conformational structure of poly(8-bromoadenylic acid) [poly(8BrA)] have been investigated using ¹H- and ¹³C-nmr, CD, and ir spectroscopy. The results have been compared with the structure of the related 5′-mono- and polynucleotides. The chemical shifts of H(2′), H(3′), C(2′), and C(3′) nmr signals show an interesting correlation with both the puckering of ribose ring and glycosidic bond torsion angle. Poly(8BrA) shows an upfield shift of the C(3′) signal and a downfield shift of the H(3′) signal compared to the chemical shifts in poly(A). These shifts are consistent with a C(3′) endo-syn conformation for poly(8BrA). A similar effect has been reported previously and is also observed here on the C(2′) and H(2′) signals when the preferred conformation is C(2′)endo-syn (e.g., in 5′-8BrAMP). The chemical-shift parameters thus act as a probe for studying syn ? anti and N ? S equilibria in solutions. The three-bond ¹H-′¹³C coupling constants between H(1′) and C(8) and C(4) have been measured in poly(8BrA) and 5′-8BrAMP and their structural implications have been discussed. The observed preference of a C(3′)endo-syn conformation for poly(8BrA), coupled with other evidence, throws doubt on the validity of a correlation previously reported whereby a syn conformation is associated with a C(2′)endo ribose pucker. The backbone conformation of randomly coiled poly(8BrA) is very similar to the structures found in polyribonucleotides: poly(A) and poly(U). All three polymers show strong preferences for the backbone angles found in RNA helices. The CD spectrum of poly(8BrA) has a striking relationship to that of poly(A). The signs of all extrema are inverted, and the magnitudes are related by a constant factor. We suggest that these differences result from a change in the angle between coupled transition moment vectors in the two polymers. Infrared spectra of poly(8BrA) in H₂O and D₂O solution are reported for the frequency range below 1400 cm^?1. The antisymmetric >PO stretching vibration is observed at an unusually low frequency in the helix (1214 cm^?1). The symmetric >PO stretch occurs at ～1095 cm^?1 but is not resolved from a ring vibration near this frequency. A conformationally sensitive band, characteristic of helical RNA structures, is observed at 817 cm^?1 and disappears when the helix is melted. This observation confirms the conclusion that ordered poly(8BrA) has a regular helical structure with an RNA backbone conformation. A stereochemical explanation is provided for the failure of poly(8BrA) (or other syn polymers) to form double helices with anti-polyribonucleotides.     

Molecular mechanics studies on poly(purine).poly(pyrimidine) sequences in DNA: polymorphism and local variability

Energy minimization has been carried out on three poly(purine).poly(pyrimidine) sequences--d(G)10.d(C)10, d(A)10.d(T)10, and d(AG)5.d(CT)5--using the molecular mechanics program AMBER (Assisted Model Building and Energy Refinement). In order to extensively scan the conformational space available, five different helical models were studied, three of them being right-handed helices while the other two were left helical. For all three sequences the right-handed A- and B-type helices are energetically slightly preferred over the left helices, but the energy difference between the various right-handed helices is only marginal. A detailed analysis has been carried out to characterize the local structural variability in the refined structures, both in terms of torsion angles as well as other parameters such as base-pair tilt, wedge roll, and wedge tilt, etc. All three sequences exhibit similar structural features for a particular form, but both the forms A and B show significant deviations from fiber models. In particular, the A-form structures have higher unit rise (2.7 A), and lower unit twist (31 degrees) and base-pair tilt (12 degrees), compared to the fiber model, which has corresponding values of 2.56 A, 32.7 degrees, and 20 degrees, respectively. All these changes indicate that the refined models are closer to the A-form structure observed in crystals of oligonucleotides. In the refined B-for models, the helical parameters are close to the fiber B-form, although the torsion angles show considerable variations. None of the three sequences examined, including the d(A)n.d(T)n sequence, show any pronounced curvature for the B-form structure.     

Interaction of transition metal ions with Z form poly d(A-C).poly d(G-T) and poly d(A-T) studied by I.R. spectroscopy.

Interactions between Ni2+, Co2+ and purine bases have been studied by I.R. spectroscopy in the case of double stranded regularly alternating purine-pyrimidine polynucleotides poly d(A-T), poly d(A-C).poly d(G-T) and poly d(G-C). The spectra of polynucleotide films have been recorded in hydration and salt content conditions which correspond to the obtention of the classical right-handed (A,B) and left-handed (Z) helical conformations. Selective deuteration of the 8C site of purines has been obtained and is used to detect interactions between the transition metal ions and the adenine or guanine bases. The spectral region between 1500 and 1250 cm-1 corresponding to base in-plane vibrations and involving also the glycosidic linkage torsion is discussed in detail. The selective interaction between the transition metal ion and the 7N site of the purine base is considered to be partly responsible for the stabilization of the base in a syn conformation, which favours the adoption by the polynucleotide (poly d(G-C), poly d(A-C).poly d(G-T) or poly d(A-T)) of a Z type conformation.     

Proton magnetic resonance and optical spectroscopic studies of watr-soluble polypeptides: poly-L-lysine HBr, poly(L-glutamic acid), and copoly(L-glutamic acid42, L-lysine HBr28, L-alanine)

The helix-coil transition has been studied by high-resolution NMR for three water-soluble polypeptides. Such systems are better models for protein behavior than those in TFA-CDCl₃ solvent. An upfield shift of ～7 cps is observed for the α-CH peak of poly(L -glutamic acid) and poly-L -lysine as the helix content increases over the transition. No such shift is found for copoly(L -glutamic acid⁴², L -lysine²⁸, L -alanine³⁰). The width of the α-CH peak for poly L-lysine increases rapidly as helix content rises but for poly L -glutamic acid and the copolymer, the width of this peak remains unchanged up to 60% helicity. This demonstrates a rapid rate of interconversion between helical and random conformations in partly helical polymer for the latter two polypeptides. All three polymers however, show no apparent α-CH peak at 100% helicity. Side-chain resonance lines also broaden as helix content increases and, to a greater extent, the closer the proton is to the main chain.     

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.     

CD studies of synthetic polypeptides as histone models: Poly(L-Arg-L-Ile-Gly), poly(L-Arg-L-Nva-Gly), and poly(L-Arg-L-Nle-Gly)

Sequential polypeptides (L -Arg-X-Gly)_n were prepared as synthetic models of arginine-rich histones to study their structure and their stereospecific interactions with DNA. In our previous work the conformational characteristics of poly(L -Arg-L -Ala-Gly), poly(L -Arg-L -Val-Gly), and poly(L -Arg-L -Leu-Gly) have already been analyzed. To obtain further insight into the influence of the X residue side chain on the conformation of the (L -Arg-X-Gly)_n polytripeptides, we now report their synthesis and cd properties when X represents the amino acid residues Ile, Nva, and Nle. The pentachlorophenyl active esters of the appropriate tripeptides were used to perform the polymerization, and the toluene-4-sulfonyl group was used to protect the arginine guanido group. CD spectroscopy showed that, in 100% trifluoroethanol, the degree of helical conformation increased in the order Ile → Nle → Nva. An equilibrium between β-turn, α-helix, and random-coil conformers occurred in 100% hexafluoroisopropyl alcohol, while a rise in the temperature or the addition of water favored the α-helix, the highest percentage of which was observed in a mixture of hexafluoroisopropyl alcohol: water (20 : 80) and in the order Ile → Nle → Nva. In aqueous solutions (at pH 7 and 12) the polymers behaved as a random coil, but they were forced to a less aperiodic structure, over a range of ionic strengths (0–0.5M NaF). A rise in temperature of up to 70°C in 100% trifluoroethanol resulted in a decrease of the α-helix percentage of the polymers, while in aqueous solutions the aperiodic structure decreased with increasing temperature. This study proved the importance of the nature of the X residue (length, C_β branching) in relation to the structural order of the sequential polypeptides. We concluded that the polymers prepared are suitable models for arginine-rich histones.     

Circular dichroism of polynucleotides: Interactions of NiCl2 with poly(dA-dT).poly(dA-dT) and poly(dG-dC).poly(dG-dC) in a water-in-oil microemulsion

The thermal behavior of the synthetic, high molecular weight, double stranded polynucleotides poly(dA-dT).poly(dA-dT) [polyAT] and poly(dG-dC).poly(dG-dC) [polyGC] solubilized in the aqueous core of the quaternary water-in-oil cationic microemulsion CTAB|n-pentanol|n-hexane|water in the presence of increasing amounts of NiCl(2) at several constant ionic strength values (NaCl) has been studied by means of circular dichroism and electronic absorption spectroscopies. In the microemulsive medium, both polynucleotides show temperature-induced modifications that markedly vary with both Ni(II) concentration and ionic strength. An increase of temperature causes denaturation of the polyAT duplex at low nickel concentrations, while more complex CD spectral modifications are observed at higher nickel concentrations and ionic strengths. By contrast, thermal denaturation is never observed for polyGC. At low Ni(II) concentrations, the increase of temperature induces conformational transitions from B-DNA to Z-DNA form, or, more precisely, to left-handed helical structures. In some cases, at higher nickel concentrations, the CD spectra suggest the presence of Z'-type forms of the polynucleotide.     

Design of mucoadhesive polymeric films based on blends of poly(acrylic acid) and (hydroxypropyl)cellulose

Mixing of aqueous solutions of poly(acrylic acid) and (hydroxypropyl)cellulose results in formation of hydrogen-bonded interpolymer complexes, which precipitate and do not allow preparation of homogeneous polymeric films by casting. In the present work the effect of pH on the complexation between poly(acrylic acid) and (hydroxypropyl)cellulose in solutions and miscibility of these polymers in solid state has been studied. The pH-induced complexation-miscibility-immiscibility transitions in the polymer mixtures have been observed. The optimal conditions for preparation of homogeneous polymeric films based on blends of these polymers have been found, and the possibility of radiation cross-linking of these materials has been demonstrated. Although the gamma-radiation treatment of solid polymeric blends was found to be inefficient, successful cross-linking was achieved by addition of N,N'-methylenebis(acrylamide). The mucoadhesive potential of both soluble and cross-linked films toward porcine buccal mucosa is evaluated. Soluble films adhered to mucosal tissues undergo dissolution within 30-110 min depending on the polymer ratio in the blend. Cross-linked films are retained on the mucosal surface for 10-40 min and then detach.     

The Specificity of Antibodies Elicited by Poly(dG)-Poly(dC)

Abstract

Antibodies have been raised to the synthetic DNA polymer poly(dG)·poly(dC). These antibodies have the ability to distinguish this right-handed polymer from natural mixed sequence DNA, as well as from other right- and left-handed synthetic DNA polymers. They show reduced but measurable binding to synthetic polymers which contain various combinations of guanine and cytosine polynucleotides suggesting that both helical shape and sequence are recognized by this antiserum.     

Conformational transition of poly(L-glutamic acid) in aqueous solution

Further direct evidences are given that a clear correlation exists between potentiometric and spectroscopic measurements in monitoring the poly(L-glutamic, acid) helix⁺ coil transtition. Specific Li⁺ ion poly (L -glUtamic acid) interactions have been observed, suresting that Li⁺ ions may exert a distinct destabilizing action on the helical conformation of the polyelectrolyte.     

Fluorescence studies on the complex formation between poly(rA) containing 1,N6-ethenoadenosine and poly(rU)

The interaction of the 1,N6-etheno derivatives of poly(rA) (poly(epsilon rA] with poly(rU) has been studied by absorption and fluorescence spectroscopy. The stoichiometry of the interaction is found to be 1 epsilon A:1 rU and 1 epsilon A:2 rU as well as in the case of poly(rA)-poly(rU) interaction. The fluorescence properties, including the intensity and polarization of fluorescence, respond to the conformational transition of poly(epsilon rA)-poly(rU) complexes. The introduction of epsilon A groups into poly(rA) results in a marked decrease in the melting temperature, suggesting that epsilon A may destabilize the helical structure. The three-exponential decay law obtained with poly(epsilon rA)-poly(rU) complexes indicates the existence of at least three different stacked conformational states.     

Vacuum UV CD of the low-salt Z-forms of poly(rG-dC).poly(rG-dC), and poly(dG-m5dC).poly(dG-m5dC)

The vacuum CD spectra of poly(rG-dC)·poly(rG-dC) and poly(dG-m⁵dC)·poly(dG-m⁵dC) have been obtained for the low-salt Z-conformations of both polymers. The spectra are very similar to those for the high-salt Z-forms. This behavior is consistent with the suggestion that the low- and high-salt Z-forms are comprised of different proportions of Z_I- and Z_II-conformations.     

The circular dichroism of poly d(AAT): d(ATT) and poly r(AAU):r(AUU). A test of the first-neighbor hypothesis

We present absorption and circular dichroism (CD) spectra for the synthetic polymers poly d(AAT):d(AAT) and poly r(AAU):r(AAU), in both native and heat-denatured forms. As a means of evaluating the first-neighbor hypothesis, the CD spectra are compared with approximations derived from spectra of other synthetic polymers containing the same first-neighbor sequences. This is the first instance where such a comparison has been possible using spectra of double-stranded RNA sequences, and the agreement between the measured and approximated spectra for poly r(AAU):r(AUU) is surprisingly good. We have also subjected the CD spectrum of poly d(AAT):d(AAT) to a previously published analytical procedure for obtaining estimates of first-neighbor frequencies. In this first independent test of the procedure, we find that the analysis does infer the existence of a majority (86%) of AA, TT, AT, and TA first neighbors but does not precisely indicate their relative proportions.