Helix-coil transition of poly-gamma-N-carbobenzoxy-L-alpha, gamma-diaminobutyrate and poly-delta-N-carbobenzoxy-L-ornithine

The helix–coil transition of poly-γ-N-carbobenzoxy-L -α,γ-diaminobutyrate (PCLB) and poly-δ-N-carbobenzoxy-L -ornithine (PCLO) in chloroform–dichloroacetic acid mixtures was followed by optical rotatory dispersion. PCLB displays a “normal” temperature-induced transition, but PCLO an “inverse” one. The thermodynamic parameters for helix formation of the two polymers were determined using the Zimm-Bragg theory. The enthalpy for adding an amide residue to a helical region, ΔH, and the initiation factor σ were ΔH = ?180 cal/mole and σ = 9.2 × 10^?5 for PCLB and ΔH = +490 cal/mole and σ = 1.9 × 10^?5 for PCLO.     

Stability of the helical conformation of random L-alanine-L-lysine copolymers in aqueous solution

The potentiometric titration of random copolymers of L -lysine and L -alanine containing 0–35% alanine was carried out. The standard free-energy change for the transition of coil to helix was calculated from the titration curve, and was treated by taking account of first-neighbor interactions. For uncharged lysine ΔG° = ?140 cal/mole, and for alanine ΔG° = ?50 cal/mole in 0.06M NaBr at 25°C, indicating that the alanine helix is thermodynamically less stable than the lysine helix. The randomness in co-polymerization was confirmed by trypsin treatment.     

Dynamics of molecular organization in agarose sulphate

Nongelling solutions of structurally regular chain segments of agarose sulphate show disorder–order and order–disorder transitions (as monitored by the temperature dependence of optical rotation) that are closely similar to the conformational changes that accompany the sol–gel and gel–sol transitions of the unsegmented polymer. The transition midpoint temperature (T_m) for formation of the ordered structure on cooling is ～25 K lower than T_m for melting. Salt-induced conformational ordering, monitored by polarimetric stopped-flow, occurs on a millisecond time scale, and follows the dynamics expected for the process 2 coil ? helix. The equilibrium constant for helix growth (s) was calculated as a function of temperature from the calorimetric enthalpy change for helix formation (ΔH_cal = ?3.0 ± 0.3 kJ per mole of disaccharide pairs in the ordered state), measured by differential scanning calorimetry. The temperature dependence of the nucleation rate constant (k_nuc), calculated from the observed second-order rate constant (k_obs) by the relationship k_obs = k_nuc(1 ? 1/s) gave the following activation parameters for nucleation of the ordered structure of agarose sulphate (1 mg mL^?1; 0.5M Me₄NCl or KCl): ΔH* = 112 ± 5 kJ mol^?1; ΔS* = 262 ± 20 J mol^?1 K^?1; ΔG*₂₉₈ = 34 ± 6 kJ mol^?1; (k_nuc)₂₉₈ = (7.5 ± 0.5) × 10⁶ dm³ mol^?1 s^?1. The endpoint of the fast relaxation process corresponds to the metastable optical rotation values observed on cooling from the fully disordered form. Subsequent slow relaxation to the true equilibrium values (i.e., coincident with those observed on heating from the fully ordered state) was monitored by conventional optical rotation measurements over several weeks and follows second-order kinetics, with rate constants of (2.25 ± 0.07) × 10^?4 and (3.10 ± 0.10) × 10^?4 dm³ mol^?1 s^?1 at 293.7 and 296.2 K, respectively. This relaxation is attributed to the sequential aggregation processes helix + helix → dimer, helix + dimer → trimer, etc., with depletion of isolated helix driving the much faster coil–helix equilibrium to completion. Light-scattering measurements above and below the temperature range of the conformational transitions indicate an average aggregate size of 2–3 helices.     

Helix–coil transition of poly-N5-(3-hydroxypropyl)-L-glutamine: Thermodynamic and statistical analysis

The thermally induced conformational changes of poly-N⁵-(3-hydroxypropyl)-L -glutamine in water and in methanol–water (3:7 v/v) have been analyzed in terms of the Lifson-Roig theory. The transitions in both solvents can be described by using v = 0.017. The thermodynamic parameters for the random coil-to-helix transition of one amino acid residue at room temperature were found to be: in water, ΔH = ? 130 cal/mole and ΔS = ? 0.45 e.u.; in methanol–water (3:7 v/v), ΔH = ? 170 cal mole and ΔS = ? 0.45 e.u. The size distribution of helical segments is broad, and the results of numerical calculations are presented for three degrees of polymerization (DP = 100, 300, and 750).     

Volume and sound velocity changes associated with coil- transition of poly(S-carboxymethyl L-cysteine) in aqueous solution

The measurements were made for the volume and the sound velocity changes (ΔV and ΔU) on titrating the sodium salt of poly (S-carboxymethyl L -cysteine) with dilute HCl. For the reaction, ? COO^? + H⁺ → ? COOH, ΔV per mole of H⁺ bound was + 12. 7 ml and +11. 4 ml in salt-free and 0. 2 M NaCl solutions, respectively. Corresponding ΔU was about ?13 cm/sec in salt-free polymer solution where 11.5 mM carboxylate ion reacts with equimolar hydrogen ion. ΔV associated with the coil-to-β transition was found to be +2. 35 ml in H₂O and +1. 90 ml in 0. 2 M NaCl per mole of amino acid residue, respectively. These values are larger than those obtained for the coil-to-helix transition of poly (L -glutamic acid). ΔU for the transition was about ?30 cm/sec in salt-free solution of polymer concentration 0.0115 mole/liter. Possible sources of ΔV and ΔU for reaction; coil → β, are (1) the formation of void volume and (2) the changes in the extent of solvation in amide linkage and in side chain.     

The enthalpy of oxidation of flavin mononucleotide

The oxidation enthalpy of reduced flavin mononucleotide at pH 7.0 in 0.2 m phosphate buffer has been studied by determining the heat associated with the reaction: FMNH₂ + 2 Fe(CN)^?3₆ ? FMN + 2 Fe(CN)^?4₆ + 2 H⁺. (a) (The quinone, semiquinone, and hydroquinone forms of FMN are represented as FMN, FMNH, and FMNH₂, respectively.) Calorimetric experiments were performed in a flow microcalorimeter which was modified to prevent sample contamination by oxygen. The enthalpy observed for reaction (a), after correction for dilution and buffer effects, was ?39.2 ± 0.4 kcal (mole FMNH₂)^?1 at 25 °C. The potential difference, ΔE′, developed by reaction (a) was determined potentiometrically and corresponded to a free energy change, ΔG′, of ?30.3 kcal (mole FMNH₂)^?1. The resulting entropy change, ΔS′, was thus calculated to be ?29.8 e.u. Reaction (a) was also studied at temperatures of 7 °C and 35.5 °C. ΔC_p′ for the reaction was calculated as ?155 ± 18 cal deg^?1 (mole FMNH₂)^?1 at 20 °C. ΔH′ for the reaction (b), FMNH₂ ? FMN + H₂, (b) was calculated as +14.2 ± 0.7 kcal mole^?1 at 25 °C, relative to the enthalpy of the hydrogen electrode being identically equal to zero at all values of pH and temperature. The free energy at pH 7.0 for reaction (b), calculated from the potential was found to be ?9.7 kcal mole^?1, which resulted in an entropy for reaction (b) of 80.2 e.u. A thermal titration of reaction (a) was used to calculate the thermodynamic parameters for the formation of semiquinone dimer according to the reaction FMNH₂ + FMN ? (·FMNH)₂. (c) The free energy, enthalpy, and entropy changes for reaction (c) were estimated to be ?6.1 kcal mole^?1, ?7 kcal mole^?1, and ?3 e.u., respectively.     

Thermal and charge-induced coil to -helix transition of poly-L-glutamic acid and random L-glutamic acid-L-alanine copolymers

Thermal and charge induced random coil to α-helix transitions of poly-L -glutamic acid (PGA) were measured by optical rotatory dispersion in various solvents. The data of PGA in 0.1M Nacl were analyzed by the Zimm-Rice theory. Enthalpy and entropy changes for the coil-to-helix transition in the unionized state were obtained: ΔH° = ?1020 ± 100 cal/residue mole; ΔH° = ?3.0 ± 0.4 e.u./residue mole. The initiation parameter, σ, of the Zimm-Rice theory was given by a value of 5 ± 1 × 10^-3. Random copolymers of L -glutamic acid and L -alanine containing 10, 30, and 40 molar percents of alanyl residue were synthesized. Stabilities of α-helix of the copolymers were compared to that of PGA. In water and water-ethanol solutions, stabilities of the polymers were almost equal after the simple correction about the ionized charge density of the polymers. In 0.1 M NaCl solution these copolymers showed some deviations from the transition curve of PGA, which would suggest the hydrophobic contribution of the alanyl residues.     

The random coil leads to beta transition of copolymers of L-lysine and L-valine: potentiometric titration and circular dichroism studies.

A series of copolymers of L -lysine and L -valine [poly(L -lysine^f L -valine^100-f)] containing 0–13% L -valine have been studied, in 0.10M KF solution, using potentiometric titration and circular dichroism spectroscopy. Incorporation of increasing amounts of valine into the copolymers favors β-sheet formation over α-helix formation at high pH and room temperature. The titrations were analyzed using the method of Zimm and Rice and the partial free energy (ΔG⁰_cβ) for the coil-to-β-sheet transition for valine is estimated at 900 cal/mole at 25°C. From the temperature dependence of the free energy, the partial enthalpy, ΔH⁰_cβ, and entropy, ΔS⁰_cβ, of the transition for valine is estimated to be 854 cal/mole and 6.0 e.u., respectively. The corresponding partial thermodynamic parameters for L -lysine are in agreement with published results. The fraction of β-sheet versus pH has been calculated for poly(L -lysine^86.8 L -valine^13.2) at 25.0°C using the titration data; data obtained from circular dichroism spectroscopy for the same copolymer are in good accord. It is concluded from these results that L -valine is a very strong β-sheet forming amino acid. Furthermore, these results indicate that the Zimm–Rice method is applicable to transitions between the coil and β-sheet states for a polypeptide containing two different residues.     

Calorimetric measurement of enthalpy change in the isothermal helix-coil transition of poly(L-ornithine) in aqueous solution.

The enthalpy change associated with the isothermal pH-induced uncharged coil-to-helix transition ΔH_h° in poly(L -ornithine) in 0.1 N KCl has been determnined calorimetrically to be ?1530 ± 210 and ?1270 ± 530 cal/mol at 10° and 25°C, respectively. Titration data provided information about the state of charge of the polymer in the calorimetric experiments, and optical rotatory dispersion data about its conformation. In order to compute ΔH_h°, the observed calorimetric heat was corrected for the heat of breaking the sample cell, the heat of dilution of HCl, the heat of neutralization of the OH^? ion, and the heat of ionization of the δ-amino group in the random coil. The latter was obtained from similar calorimetric measurements on poly(D ,L -ornithine). Since it was discovered that poly(L -ornithine) undergoes chain cleavage at high pH, the calorimetric measurements were carried out under conditions where no degradation occurred. From the thermally induced uncharged helix–coil transition curve for poly(L -ornithine) at pH 11.68 in 0.1 N KCl in the 0°–40°C region, the transition temperature T_tr and the quantity (?θ_h/?T)_Ttr have been obtained. From these values, together with the measured values of ΔH_h°, the changes in the standard free energy ΔG_h° and entropy ΔG_h°, associated with the uncharged coil-to-helix transition at 10°C have been calculated to be ?33 cal/mol and ?5.3 cal/mol deg, respectively. The value of the Zimm–Bragg helix–coil stability constant σ has been calculated to be 1.4 × 10^?2 and the value of s calculated to be 1.06 at 10°C, and between 0.60 and 0.92 at 25°C.     

Syntheses and conformational studies of poly(Nε-methyl-L-lysine), poly(Nδ-methyl-L-ornithine), poly(Nδ-ethyl-L-ornithine), and their carbobenzoxy derivatives

The helix–coil transitions of poly(N^ε-methyl, N^ε-carbobenzoxy-L -lysine), poly(N^δ-methyl, N^δ-carbobenzoxy-L -ornithine), and poly(N^δ-ethyl, N^δ-carbobenzoxy-L -ornithine) in chloroform–dichloroacetic acid and their corresponding decarbobenzoxylated polypeptides in alkaline solutions were followed by optical rotation measurements. The introduction of a methyl or an ethyl group to the side chains of the carbobenzoxy derivatives of poly(L -lysine) and poly(L -ornithine) appeared to weaken the helical conformation at 25°C. The thermodynamic quantities of the three water-soluble polypeptides were calculated from the data on potentiometric titrations at several temperatures. For uncharged coil-to-helix transition, ΔH = ?370 cal/mol and ΔS = ?1.1 eu/mol for poly(N^ε-methyl-L -lysine), and ΔH = ?540 cal/mol and ΔS = ?1.6 eu/mol for poly(N^δ-ethyl-L -ornithine) (all on molar residue basis). The absolute values of ΔH and ΔS dropped in the region of pH-induced transition and eventually both quantities became positive. The initiation factor σ was about 2 × 10^?3, which was essentially independent of temperature. For poly(N^δ-methyl-L -ornithine) the coil-to-helix transition was not complete even when the polymer was uncharged at high pH.     

Conformational studies of poly-L-alanine in water

The conformational properties of poly-L -alanine have been examined in aqueous solutions in order to investigate the influence of hydrophobic interactions on the helix–random coil transition. Since water is a poor solvent for poly-L -alanine, water-soluble copolymers of the type (D , L -lysine)_m–(L alanine)_n-(D , L -lysine)_m, having 10, 160, 450, and 1000 alanyl residues, respectively, in the central block, were synthezised. The optical rotatory dispersion of the samples was investigated in the range 190–500 mμ, and the rotation at 231 mμ was related to the α-helix content, θ_H, of the alanine section. In salt-free solutions, at neutral pH, the three large polymers show high θ_H values, which are greatly reduced when the temperature is increased from 5 to 80°C. No helicity was observed for the small (n = 10) polymer. By applying the Lifson-Roig theory, the following parameters were obtained for the transition of a residue from a coil to a helical state: ν = 0.012; ΔH = ?190 ± 40 cal./mole; ΔS = ?0.55 ± 0.12 e.u. Since ΔH and ΔS differ from the values expected for a process involving only the formation of a hydrogen bond, and in a manner predicted by theories for the influence of hydrophobic bonding on helix stability, it is concluded that a hydrophobic interaction is also involved. In the presence of salt (0.2M NaCl), or when the ε-amino groups of the lysyl residues are not protonated (pH = 12), the helical form of the two large polymers (n = 450 and n = 1000) is more stable than in water. Since the electrostatic repulsion between the lysine end blocks is greatly reduced under these conditions, the alanine helical sections fold back on themselves, and this conformation is stabilized by interchain hydrophobia bonds. This structure was predicted by the theory for the equilibrium between such interacting helices, non-interacting helices, and the random coil.     

Adsorption Optimization of Lead (II) Using Saccharum Bengalense as a Non-Conventional Low Cost Biosorbent: Isotherm and Thermodynamics Modeling

In the present study a novel biomass, derived from the pulp of Saccharum bengalense, was used as an adsorbent material for the removal of Pb (II) ions from aqueous solution. After 50 minutes contact time, almost 92% lead removal was possible at pH 6.0 under batch test conditions. The experimental data was analyzed using Langmuir, Freundlich, Timken and Dubinin-Radushkevich two parameters isotherm model, three parameters Redlich—Peterson, Sip and Toth models and four parameters Fritz Schlunder isotherm models. Langmuir, Redlich—Peterson and Fritz-Schlunder models were found to be the best fit models. Kinetic studies revealed that the sorption process was well explained with pseudo second-order kinetic model. Thermodynamic parameters including free energy change (ΔG°), enthalpy change (ΔH°) and entropy change (ΔS°) have been calculated and reveal the spontaneous, endothermic and feasible nature of the adsorption process. The thermodynamic parameters of activation (ΔG ^#, ΔH ^#and ΔS ^#) were calculated from the pseudo-second order rate constant by using the Eyring equation. Results showed that Pb (II) adsorption onto SB is an associated mechanism and the reorientation step is entropy controlled.     

Cis-Trans equilibrium and kinetic studies of acetyl-L-proline and glycyl-L-proline

By means of carbon-13 nmr (at 25 MHz) the trans/cis conformer ratio in glycyl-L -proline has been measured in aqueous (D₂O) solution over the temperature range 33–96°C. It is found that ΔH⁰ = ?4.2 kJ/mole and ΔS⁰ = ?9.7 J/mole/K. Measurements of the T₁ values for the proline ring carbons yielded values consistent with a fast puckering process involving both the β- and γ-carbons. Measurements of the rate of cis-trans conformational interconversion in glycyl-L -proline, using complete line-shape analysis for the glycyl α-carbon resonance, gave values for the trans → cis isomerization as follows: ΔH^≠ = 83.5 ± 0.2 kJ/mole; ΔS^≠ = 0.0 ± 10 J/mole/K. A more approximate determination from coalescence temperature observations gave a value of ΔG^≠ of 82.0 ± 0.4 kJ/mole for this process in acetyl-L -proline in aqueous solution. The presence of 12M NaSCN lowered this barrier by ca. 2.6 kJ/mole. Such measurements are relevant to present theoretical models of the denaturation-renaturation processes in proteins, in which proline residues may play a key role.     

Thermodynamics of the partition of chondroitin sulfate–hexadecylpyridinium complexes in butanol/aqueous salt biphasic solutions

The thermodynamics of the partition of chondroitin sulfate–hexadecylpyridinium complexes wee studied in order to gain further insight into the mechanisms responsible for the sensitivity of the relative solubility of these complexes in aqueous slat and butanol phases to small changes in slat concentration. The dependence of the partition coefficient was measured as a function of temperature at three different salt concentrations. Increasing the temperature was found to favor the form of the complex which was soluble n the aqueous phase. Although the transition could be induced by temperature changes, he transition occurred over a 20°C range in temperature. The transition from the aqueous phase to the butanol phase was strongly exothermic, with ΔH = ?22.3 kcal/mol polymer. The value of ΔS was found to be dependent on the salt concentration, ranging from ?72.7 e.u./mol polymer in 0.05125M NaCl to ?77.1 e.u./mol polymer in 0.05375M MaCl. When placed on a disaccharide basis, the corresponding values are ΔH = ?402 cal/mol and ΔS = ?1.31 to ?1.3 e.u./mol. The sharpness of he transition was found to be due t the similarity in magnitude of ΔH and TΔS, and on the dependence of the later upon the salt concentration.     

The random coil → β transition of copolymers of L-lysine and L-isoleucine: Potentiometric titration and circular dichroism studies

Copolymers of L -lysine and L -isoleucine [poly(L -Lys^f,L -Val^{1 ? f})] containing 4–15% isoleucine were investigated using potentiometric titration and circular dichroism (CD) spectroscopy. With increasing isoleucine content, β-sheet formation is favored over α-helix formation at high pH and room temperature. The fraction of β-sheet present, as a function of pH, calculated from titrations of poly(L -Lys^85.2,L -Ile^14.8), agreed well with data obtained from CD studies for the same copolymer. Thermodynamic parameters were determined from titrations using the method of Zimm and Rice; the partial free energy (ΔG°_{C → β}) at 25° for the coil-to-β-sheet transition for isoleucine was estimated to be ?515 cal/mol; from the temperature dependence of free energy, the partial entropy (ΔS°_cβ), and the partial free enthalpy (ΔH°_{c → β}) of the coil → β transition for isoleucine is estimated to be 2.6 e.u. and 260 cal/mol, respectively. The partial thermodynamic parameters obtained for lysine are in good agreement with literature values. It is concluded from these studies that isoleucine has a very high potential for a β-sheet formation.     

Thermodynamics of equilibria of hemoglobins M Milwaukee-I and Saskatoon and isolated chains of hemoglobin a with carbon monoxide

The thermodynamic parameters of the CO-equilibria of isolated chains of hemoglobin A and of two α-chains in hemoglobins M Milwaukee-I and Saskatoon at 25°, pH 7.0 were determined. The parameters for the binding of the first CO molecule to the hemoglobins M were ΔH′=?17 and ?18 kcal/mole heme and ΔS′=?30 and ?29 e.u. for hemoglobins M Milwaukee-I and Saskatoon, respectively. In contrast to this the characteristics of the second step of the binding were ΔH′=+5.9^· and +4.3 kcal/mole and ΔS′=+51 and +49 e.u. These values for the second step were also significantly different from those of the isolated α-chain (ΔH′=?15 kcal/mole and ΔS′=?11 e.u.).     

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..     

A study of the growth of normal and iodinated collagen fibrils in vitro using electron microscope autoradiography

Electron microscopic autoradiographic observations on collagen fibrils grown in vitro allow growth rates in the N- and C-terminal directions to be measured on individual fibrils. Such observations, made on normal and iodinated collagen, show that normal fibrils grow at both ends (although rather more rapidly at the N-terminal end), whereas fully-iodinated collagen fibrils grow only at the N-terminal end. Measurements of growth rates at different temperatures provide estimates of the activation enthalpy (ΔH^≠) and entropy (ΔS^≠) of precipitation for the two types of collagen. Solubility measurements have also yielded values for the thermodynamic enthalpy (ΔH) and entropy (ΔS) of precipitation. Results show that the activated (rate-limiting) state is characterized by a large positive ΔH^≠ and ΔS^≠ similar in magnitude to the ΔH and ΔS of transition from solution to fibril. It is also concluded that the different rates of precipitation of normal and iodinated collagen cannot be explained in terms of fibril formation requiring ionization of the tyrosine residues.     

Design of Peptides Using α,β-Dehydro-residues: Synthesis,Crystal Structure and Molecular Conformation of N-Boc-L-Val-ΔPhe–ΔPhe-L-Ala-OCH3

To obtain general rules of peptide design using α,β-dehydro-residues, a sequence with two consecutive ΔPhe-residues, Boc-L -Val-ΔPhe–ΔPhe- L -Ala-OCH₃, was synthesized by azlactone method in solution phase. The peptide was crystallized from its solution in an acetone/water mixture (70:30) in space group P6₁ with a=b=14.912(3) Å, c= 25.548(5) Å, V=4912.0(6) ų. The structure was determined by direct methods and refined by a full matrix least-squares procedure to an R value of 0.079 for 2891 observed [I?3σ(I)] reflections. The backbone torsion angles ?₁=?54(1)°, ψ₁= 129(1)°, ω₁=?177(1)°, ?₂ =57(1)°, ψ₂=15(1)°, ω₂ =?170(1)°, ?₃=80(1)°, ψ₃ =7(2)°, ω₃=?177(1)°, ?₄ =?108(1)° and ψ^T₄=?34 (1)° suggest that the peptide adopts a folded conformation with two overlapping β-turns of types II and III′. These turns are stabilized by two intramolecular hydrogen bonds between the CO of the Boc group and the NH of ΔPhe³ and the CO of Val¹ and the NH of Ala⁴. The torsion angles of ΔPhe² and ΔPhe³ side chains are similar and indicate that the two ΔPhe residues are essentially planar. The folded molecules form head-to- tail intermolecular hydrogen bonds giving rise to continuous helical columns which run parallel to the c-axis. This structure established the formation of two β-turns of types II and III′ respectively for sequences containing two consecutive ΔPhe residues at (i+2) and (i+3) positions with a branched β-carbon residue at one end of the tetrapeptide.     

A Raman and calorimetric investigation of 3′, 5′ GpG

The Raman spectra of guanylyl (3′-5′) guanosine (GpG) in solution in H₂O and D₂O at pH 3–7 have been recorded at various temperatures between 0 and 80°C. The results are consistent with the existence in the lower temperature range of stable aggregates formed by the stacking of GpG tetramers. The aggregates melt cooperatively near 60°C, which results in important changes in the spectra. Among these, a large increase in intensity of some of the bands assigned to the guanine residues shows that unstacking of the bases occurs at the melting. Also apparent in the spectra are changes in the intensity and frequency of band attributable to molecular groups involved in intermolecular hydrogen bonding between adjacent molecules in the complex. The melting temperature of GpG decreases by approximately 15°C upon lowering the concentration from 5 × 10^?2 to 5 × 10^?4M, as shown by Raman, calorimetric, CD, and uv measurements. The experimentally determined ΔH and ΔS for the melting transition are 9 Kcal/mol and 28 e.u./mol, respectively. The aggregation of GpG in 1.5 × 10^?3M solutions was found to be very slow. The half-time of the process, which roughly follows first-order kinetics, is approximately 3 min at 10°C and 21 min at 35°C. The negative energy of activation associated with this reaction (?143 Kcal) indicated that the process involves intermediates whose concentrations decrease the temperatures raised, thus slowing down the overall process. The rate of disaggregation of GpG upon dilution to very low concentration is also extremely slow, indicating that the GpG aggregates, once formed, are very stable.