Protonated polynucleotide structures. IX. Disproportionation of poly (G)-poly (C) in acid medium

The interaction between poly (G) and poly (C) was investigated in neutral and acid medium by optical methods. Three main points arise from this investigation. (1) The formation of poly (G)·poly (C) was complete only above an ionic strength of about 0.6M [Na⁺]. Lowering the ionic strength increased the amounts of free poly (G) and free poly (C) that could be detected. (2) When titrating towards acid pH values a transition took place which was characterized by potentiometry, mixing curves, and circular dichroism: a three-stranded poly (G)·poly (C)·poly (C⁺) complex was formed analogous to the transition observed for the acid titration of poly (I)·poly (C). (3) Even when the poly (G)·poly (C) complex was incompletely formed (at low ionic strength) in neutral medium all poly (C) entered the triple-stranded complex.     

Experimental thermodynamics of the helix--random coil transition. 3. Determination of the transition enthalpies of the helical complexes poly(I+C) and poly I in solution

The enthalpies of the helix-coil transitions of the ordered polynucleotide systems of poly(inosinic acid)–poly(cytidylic acid) [poly(I + C)], (helical duplex), and of poly (inosinic acid) [poly(I + I + I)], (proposed secondary structure: a triple-stranded helical complex), were determined by using an adiabatic twin-vessel differential calorimeter. Measuring the temperature course of the heat capacity of the aqueous polymer solutions, the enthalpy values for the dissociation of the helical duplex poly (I + C) and the three-stranded helical complex poly(I + 1 + 1), respectively, were obtained by evaluating the additional heat capacity involved in the conformational change of the polynucleotide system in the transition range. The ΔH values of the helix-coil transition of poly (I + C) resulting from the analysis of the calorimetric measurements vary between the limits 6.5 ± 0.4 kcal/mole (I + C) and 8.4 ± 0.4 kcal/mole (I + C). depending on the variation of the cation concentration ranging from 0.063 mole cations kg H₂O to 1.003 mole cations/kg H₂O. The calorimetric investigation of an aqueous poly I solution (cation concentration 1.0 mole/kg H₂O) yielded the enthalpy value ΔH = 1.9 ± 0.4 kcal/mole (I), a result which has been interpreted qualitatively following current models of inter- and intramolecular forces of biologically significant macromolecules. Additional information on the transition behavior of poly(I+ C)Was obtained by ultraviolet and infrared absorption measurements.     

Polynucleotides. XLIV. Synthesis and properties of poly (2-azaadenylic acid) and poly(2-azainosinic acid).

Chemically synthesized 2-azaadenosine 5'-diphosphate (n2ADP) and 2-azainosine 5'-diphosphate (n2IDP) were polymerized to yield poly(2-azaadenylic acid), poly(n2A), and poly(2-azainosinic acid), poly(n2I), using Escherichia coli polynucleotide phosphorylase. In neutral solution, poly(n2A) and poly(n2I) had hypochromicities of 32 and 5.5%, respectively. Poly(n2A) formed an ordered structure, which had a melting temperature (Rm) of 20 degrees C at 0.15 M salt concentration. Upon mixing with poly(U), poly(n2A) formed a 1 : 2 complex with Tm of 41 degrees C at 0.15 M salt concentration. Poly(n2A) and poly(n2I) formed three-stranded complexes with poly(I), and poly(A), respectively. Poly(n2A) . 2poly(I), poly(A) . 2poly(n2I), and poly(n2A) . 2poly(n2I) complexes had Tm values of 23, 48, and 31 degrees C at 0.15 M salt concentration, respectively. Poly(n2I) formed a double-stranded complex with poly(C), but its Tm was very low.     

An X-ray diffraction study of poly-L-homoarginine hydrochloride.

An X-ray study of the synthetic polypeptide poly(L -homoarginine hydrochloride) has been made to investigate whether, like the chemically related polypeptides poly(L -lysine hydrochloride), poly(L -arginine hydrochloride), and poly(L -ornithine hydrobromide), it can undergo conformational transitions merely from variations in its degree of hydration. X-ray photographs of powder and oriented specimens containing one to 15 molecules of water per L -homoarginine hydrochloride residue showed that this polymer forms only a β-pleated-sheet structure. The pleated sheets, formed by antiparallel polypeptide chains hydrogen-bonded to each other, are piled up along the b axis in an alternating sequence (“sandwich structure”). This structure did not appreciably change with variations of the degree of hydration, and the observed reflections at 56% relative humidity (1.8 molecules of water per residue) could be indexed satisfactorily in terms of a monoclinic unit cell, of space group P2₁, with a = 9.34 Å, b = 40.07 Å, c = 6.94 Å, and γ = 106°. These dimensions are shown by models to be compatible with the proposed structure, and the calculated density of 1.27 g/cm³ agrees well with the experimental value of 1.29 g/cm³. Removal of the last molecule of water results in a very diffuse pattern, while specimens containing 20 molecules of water per residue show only reflections due to water.     

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.     

Estimation of amino acid residue side-chain absorption in the infrared spectra of protein solutions in heavy water.

The values of maximum frequencies, intensities, and other spectral parameters of the main absorption bands of amino acid residue side-chain groups have been obtained in the 1500–1800-cm^?1 region for solutions in heavy water at pD 1–12. It is shown that absorption of residues of asparagine, glutamine, aspartic and glutamic acids, arginine, and tyrosine must be taken into account in quantitative studies of the infrared spectra of polypeptide and protein solutions in heavy water. Examples of separating out the amide I band for ribonuclease A in heavy water are given.     

A vibrational spectroscopic study of the self-association of polyinosinic acid and polyguanylic acid in aqueous solution

We have studied by Raman and ir spectroscopy the structure of self-associated polyinosinic acid and polyguanylic acid in aqueous solution. The results are consistent with the formation of a four-stranded complex, which melts cooperatively near 60°C in the case of poly (I) in the presence of K⁺ ions. The conformation of the ribose in both systems is mixed C2′-endo/C3′-endo, giving a structure that is intermediate between the extremes proposed previously from x-ray diffraction studies. Characteristic Raman bands for the C2′-endo ribose conformation in polyribonucleotides are identified. The four-stranded structure of poly (I) appears to be very flexible, with ≈15% of the tetrameric segments being disrupted and ≈30% of the ribose units adopting a disordered conformation prior to melting. This disordering process increases to ≈75% above the melting transition, with the remaining ≈25% of the ribose units keeping an ordered C2′-endo or C3′-endo conformation. © 1994 John Wiley & Sons, Inc.     

Steric structure of L-proline oligopeptides. I. Infrared absorption spectra of the oligopeptides and poly-L-proline

Poly-L -prolines I and II were differentiated by the characteristic bands in the far infrared region. Form I showed two broad bands at about 280 and 160 cm^?1 and form II two bands at, 400 and 670 cm.^?1. Furthermore, three broad bands at about 250, 200, and 100 cm.^?1 were observed in the spectrum for form II. Infrared absorption bands of the pentamer, hexamer, and octamer of tert-amyloxycarbonyl-L -proline were almost similar to those of poly-L -proline II in the 1800–75 cm.^?1 region. In the far-infrared region, especially, the absorption bands of these three oligopeptides were in good agreement with that of poly–L –proline II. Accordingly we concluded that the molecules of pentamer, hexamer, and octamer had a helical structure of a left-handed threefold screw axis. The tetrapeptide of tert-amyloxycarbonyl-L -proline might also have a left-handed helix, probably one turn, since the tetramer clearly showed an absorption band at about 400 cm.^?-1 characteristic of poly–L –proline II.     

Poly(rA) • Poly(rU) with Ni2+ Ions at Different Temperatures: Infrared Absorption and Vibrational Circular Dichroism Spectroscopy

Abstract

Phase transitions were studied of the sodium salt of poly(rA) ?poly(rU) induced by elevated temperature without Ni²⁺ and with Ni²⁺ in 0.07 M concentration in D₂O (～0.4 [Ni]/[P]). The temperature was varied from 20° C to 90° C. The double-stranded conformation of poly(rA)?poly(rU) was observed at room temperature (20° C—23° C) with and without Ni²⁺ ions. In the absence of Ni²⁺ ions, partial double- to triple-strand transition of poly(rA) ?poly(rU) occurred at 58° C, whereas only single-stranded molecules existed at 70° C. While poly(rU) did not display significant helical structure, poly(rA) still maintained some helicity at this temperature. Ni²⁺ ions significantly stabilized the triple-helical structure. The temperature range of the stable triple-helix was between 45° C and 70° C with maximum stability around 53° C. Triple-to single-stranded transition of poly(rA) ?poly(rU) occurred around 72° C with loss of base stacking in single-stranded molecules. Stacked or aggregated structures of poly(rA) formed around 86° C. Hysteresis took place in the presence of Ni²⁺ during the reverse transition from the triple-stranded to the double-stranded form upon cooling. Reverse Hoogsteen type of hydrogen-bonding of the third strand in the triplex was suggested to be the most probable model for the triple-helical structure. VCD spectroscopy demonstrated significant advantages over infrared absorption or the related electronic CD spectroscopy.     

Interaction of poly-5-bromouridylic acid. I. Formation of helical complexes with various adenine and 2-aminoadenine derivatives

Complexes of poly(BU) with various adenine derivatives were investigated by circular dichroism (CD) and absorption spectroscopy. A 1:2 stoichiometry was indicated on CD mixing curves for typical complexes of 9-substituted adenine and 2-aminoadenine derivatives with poly(BU). The CD spectrum of adenosine·2poly(BU) is characterized by well-resolved bands in the range of 210–350 nm. Other adenine derivative–poly(BU) complexes also afford similar CD spectra, while 2-aminoadenine derivative–poly(BU) complexes give quite different spectra. Attempts to assign representative CD spectra were made using the transition of helical poly(BU) and the respective purine polynucleotides. The similarity of the CD spectra suggests that poly(A)·2poly(BU) and adenine derivative–poly(BU) complexes are nearly identical in structure except for the ribose–phosphate linkage. The fact that the uv isosbestic point of adenosine·2poly(BU) falls in close proximity to that of the corresponding polymer complex also supports this conclusion. In the formation of stable helices, the ribose moiety is dispensable in the “strand” of purine. The T_m of 9-methyladenine·2poly(BU) is somewhat higher than that of adenosine·2poly(BU) under equivalent conditions. The T_m difference with the monomer–poly(U) system was found to be about 20°C in 0.4M NaCl–0.02M Na–cacodylate–5 × 10^?4M EDTA (pH 7.0). Further, it was noted that the monomer–poly(BU) complexes are formed even when the T_m is lower than that of self-folded poly(BU).     

Quantitative IR spectrophotometry of peptide compounds in water (H2O) solutions. II. Amide absorption bands of polypeptides and fibrous proteins in alpha-, beta-, and random coil conformations.

Infrared spectra of poly(D,L-alanine), poly(L-glutamic acid), poly(L-lysine), silk fibroin, and tropomyosin have been registered for various conformations of the polypeptide chain. Assuming additivity of the main- and side-chain absorption, spectral parameters of amide I and II absorption bands corresponding to alpha-, beta-, and random coil conformations have been derived. The amide I band parameters for H2O and D2O have been compared.     

Polynucleotides. LII1 Synthesis and properties of poly(2′-deoxy-2′-fluoroadenylic acid)

2′-Deoxy-2′-fluoroadenosine was chemically transformed to its 5′-diphosphate and polymerized with polynucleotide phosphorylase to give poly(2′-deoxy-2′-fluoroadenylic acid) [poly(Af)]. Polymerization proceeded smoothly as in the case of poly(A) and the yield of the polymerization was 55%. The UV absorption spectra of poly(Af) closely resembled those of poly(A) and the hypochromicity was 32% at pH 7.0. The CD profile at 25° and neutrality showed similar pattern to that of other poly(2′-deoxy-2′-halogenoadenylic acids) with somewhat larger [θ] values both in the positive and negative maxima. Acid titration of poly(Af) showed a transition point at pH 5.2 and the Tm of the acid form was 37° which was significantly lower than that of poly(A), but similar to that of poly(2′-azido-2′-deoxyadenylic acid). Poly(Af) formed 1:1 and 1:2 complexes with poly-(U) having Tm of 49° and 62° at 0.04M and 0.15M Na⁺ concentration, respectively. Poly(Af) also formed a 1:2 complex with poly(I) and its Tm was 36° at 0.05M Na⁺ concentration. These data showed that poly(Af) has rather similar properties to those of poly(A), but not to poly(dA).     

Secondary structure of peptides: 8. 13C n.m.r. CP/MAS investigation of solid poly(l-leucines) and poly(d-norvalines) prepared from N-carboxyanhydrides

¹³C n.m.r. CP/MAS spectra (50.3 and 75.4 MHz) of solid poly(l-lleucines) and poly(d-norvalines) measured with suitable acquisition parameters allow quantification of the composition of the secondary structure. The optimum acquisition parameters were found by systematic variation of the contact time by means of samples containing 5?0% α-helix structure. The polypeptides were prepared by primary or tertiary amine-initiated polymerizations of the corresponding amino acid NCAs and the average degrees of polymerization ($\text{DP}$) were determined by ¹H n.m.r. endgroup analysis. The mole fraction of α-helices increases with increasing $\text{DP}$; it depends on the nature of the solvent and to a lesser degree on the polymerization temperature. When prepared under identical conditions, poly(d-norvaline) samples contain more β-sheet structure than poly(l-leucine. Reprecipitation increases the α-helix content, demonstrating that a part of the original β-sheet structure is thermodynamically unstable. The presence of oligomers of $\text{DP}$ ?10 is mainly responsible for the thermodynamically stable part of the β-sheet structure. The chain growth mechanism is discussed.     

Analysis of histone messenger RNA of Drosophila melanogaster by two-dimensional gel electrophoresis.

The kinetics of the hydrogen-deuterium exchange reactions of double-helical poly (rI) · poly (rC), single-stranded poly(rC) and poly(rI), inosine, and cytosine- 5′-phosphoric acid have been examined, at various temperatures in the range 20 °C to 52 °C, by stopped-flow ultraviolet spectrophotometry, in the region 270 to 300 nm. For the solution of double-helical poly(rI) · poly(rC), two first-order deuteration reactions were found: a fast one and a slow one. At 25 °C and at pH 7.0, the rate constant was 12.3 s^?1 for the fast reaction, and 0.13 s^?1 for the slow reaction. The rate constant of the fast reaction is nearly equal to that of the single-stranded poly(rC) (12.6 s^?1), and is assigned to the deuteration at the amino hydrogen (that is, free from the C · I hydrogen bond) of the cytosine residue. The slow reaction is attributable to the deuteration of the two hydrogens: the amino hydrogen of rC and imide hydrogen of rI, which are rapidly exchanging with each other within every rC · rI base-pair. From the observed temperature effect on this slow reaction rate, it has been concluded that there are two types of “opening process” that are relevant to the hydrogen exchange reaction; one of them is predominent in the range 47 °C to 52 °C and the other in the temperature region lower than 47 °C. The enthalpy (H) and entropy (S) differences of the “open” and “closed” forms in the former type process are ΔH = 167 kcal per mole and ΔS = 507 e.u., while in the latter ΔH = 8.1 kcal per mole and ΔS = 10 e.u..     

Conformational properties of the sequential polyproline analogs poly(Pro-Aze-Pro) and poly(Aze-Pro-Aze)

The lack of the positive band at around 226 nm in the CD spectra of poly(prolyl-azetidine-2-carbonyl-proline) in trifluoroethanol and of poly(azetidine-2-carbonyl-prolyl-azetidine-2-carboxylic) acid in F₃EtOH and water, the hyperchromism of the absorption maximum at about 202 nm, and the extremely small intensity of the C^β-Pro, C^γ-Pro, and C^β-Aze signals for the cis peptide bonds in the ¹³C nmr spectrum of poly(Pro-Aze-Pro) in F₃EtOH indicate that both polyproline analogs exist as disordered chains in this solvent, the trans peptide group being maintained. The disordering of the chains is attributed to an increase in the accessible range of ψ due to the reduced dimensions of the square ring of L -azetidine-2-carboxylic acid residue relative to the pyrrolidine ring of proline and to strong interactions of the haloalcohol with the peptide groups of the chains.     

Protonated polynucleotides. VII. Thermal transitions between different complexes of polyinosinic acid and polycytidylic acid in an acid medium

The interaction between poly (I) and poly (C) in acid medium has been studied by potentiometric titration, mixing curves and thermal denaturation. Phase diagramms as a function of ionic strength, pH, and temperature have been established. From these data it is shown that the acid titration of the complex poly (I) · poly (C) passes through a triple-stranded intermediate poly (I) · poly (C) · poly (C⁺) to yield finally the protonated double-helical complex poly (I) · poly (C⁺). The mixing curves indicate the sole presence of the three-stranded complex in the intermediate zone. On the basis of the pK's the coexistence between the three-stranded complex with the neighboring double-stranded structure is demonstrated in a narrow rang of pH and ionic strength. The geometry of the base arrangements, their conformation and the sense of the strands are discussed in the light of the data presented. A Hoogsteen-type pairing between the bases for poly (I) · poly (C⁺) is favored, although the reverse Hoogsteen pair cannot be excluded.     

Raman spectral studies of nucleic acids. XVI. Structures of polyribocytidylic acid in aqueous solution

Raman spectra of polyribocytidylic acid show the formation of an ordered single-stranded structure [poly(rC)] at neutral pH and an ordered double-stranded structure containing hemiprotonated bases [poly(rC)·poly(rC⁺)] in the range 5.5 > pH > 3.7. Below 40°C, poly(rC) contains stacked bases and a backbone geometry of the A-type, both of which are gradually eliminated by increasing the temperature to 90°C. Below 80°C, poly(rC)·poly(rC⁺) contains bases which are hydrogen bonded and stacked and a backbone geometry also of the A-type. In this structure the bases of each strand are shown to be structurally identical, i.e., hemiprotonated, and therefore distinct from both neutral and protonated cytosines. Infrared and Raman spectra indicate the existence of a center of symmetry with respect to the paired cytosine residues, which suggests that the additional proton per base pair is shared equally by the two hydrogen-bonded bases. Denaturation of poly(rC)·poly(rC⁺) occurs cooperatively (t_m ≈ 80°C) with elimination of base stacking, base pairing, and the A-helix geometry. Each of the separated strands of the denatured complex is shown to contain comparable amounts of both neutral and protonated cytosines, most likely in alternating sequence [poly(rC, rC⁺)]. In both poly(rC, rC⁺) and poly(rC), at 90°C, the backbones do not exhibit the phosphodiester Raman frequencies characteristic of other disordered polyribonucleotide chains. This is interpreted to mean that the single strands, though devoid of base stacking and A-type structure, contain uniformly ordered backbones of a specific type. Fully protonated poly(rC⁺), on the other hand, forms no ordered structure and may be characterized as a disordered (random chain) polynucleotide at all temperatures. Several Raman lines of poly(rC) are absent from the spectrum of poly(rC)·poly(rC⁺) and vice versa. These frequencies, assigned mainly to vibrations of the ribose groups, suggest that the furanose ring conformations are different in the single-stranded and double-stranded structures of polyribocytidylic acid. Several other Raman group frequencies have been identified and correlated with the polymer secondary structures.     

The conformation of polycytidylic acid, polyguanylic acid, polyinosinic acid, and their helical complexes in aqueous solution from laser Raman scattering

The Raman spectra of the double helical complexes of poly C–poly G and poly I–poly C at neutral p^H are presented and compared with the spectra of the constituent homopolymers. When a completely double-helical structure is formed in solution a strong sharp band at 810–814 cm^?1 appears which has previously been shown to be due to the A-type conformation of the sugar–phosphate backbone chain. By taking the ratio of the intensity of the 810–814 cm^?1 band to the intensity of the 1090–1100 cm^?1 phosphate vibration, one can obtain an estimate of the fraction of the backbone chain in the A-type conformation for both double-stranded helices and self-stacked single chains. This type of information can apparently only be obtained by Raman spectroscopy. In addition, other significant changes in Raman intensities and frequencies have been observed and tabulated: (1) the Raman intensity of certain of the ring vibrations of guanine and hypoxanthine bases decrease as these bases become increasingly stacked (Raman hypochromism), (2) the Raman band at 1464 cm^?1 in poly I is asigned to the amide II band of the cis-amide group of the hypoxanthine base. It shifts in frequency upon base pairing to 1484 cm^?1, thus permitting the determination of the fraction of I–C pairs formed.     

Absorption and circular dichroism spectra of CuCl2 complexed with poly(S-carboxymethyl-L-cysteine) and poly(S-carboxyethyl-L-cysteine)

The interaction of CuCl₂ with poly(S-carboxymethyl-L -cysteine) (poly[Cys(CH₂COOH)]) and poly(S-carboxyethyl-L -cysteine) (poly[Cys(C₂H₄COOH)]) were studied by absorption spectra and circular dichroism (CD). On mixing CuCl₂ with polypeptide solutions, absorption bands appeared at 320–325 nm in both polypeptides, and at 255–260 nm in the case of poly[Cys(CH₂COOH)]. A stable bound species was formed in the case of poly[Cys(CH₂COOH)], since the apparent molar absorption coefficient of the bound species did not depend on the mixing ratio. From the absorption data, it was inferred that Cu²⁺ ions were complexed with the side chains, most probably with sulfur atoms and carboxyl groups. Induced optical activities were observed for the two polypeptides. The CD spectra of poly[Cys(CH₂COOH)] + CuCl₂ gave simpler aspects than those of poly[Cys(C₂H₄COOH)] + CuCl₂.     

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.