The hydrogen bonding of cytosine with guanine: calorimetric and 1H-nmr analysis of the molecular interactions of nucleic acid bases

The enthalpy of hydrogen-bond formation between guanine (G) and cytosine (C) in o-dichlorobenzene and in chloroform at 25°C has been determined by direct calorimetric measurement. We derivatized 2′-deoxyguanosine and 2′-deoxycytidine at the 5′- and 3′-hydroxyls with triisopropylsilyl groups; these groups increase the solubility of the nucleic acid bases in nonaqueous solvents. Such derivatization also prevents the ribose hydroxyls from forming hydrogen bonds. Consequently, hydrogen-bond formation in our system is primarily between the bases, and to a lesser extent, between base and solvent, and can be measured directly with calorimetry. To obtain the data on base-pair formation, we first took into account the contributions from self-association of each base, and where possible, have determined the ΔH of self-association. From isoperibolic titration calorimetry, our measured ΔH of C₂ formation in chloroform is ?1.7 kcal/mol of C. Our measured ΔH of C:G base-pair formation in o-dichlorobenzene is ?6.65 ± 0.32 kcal/mol. Since o-dichlorobenzene does not form hydrogen bonds, the ΔH of C:G base-pair formation in this solvent represents the ΔH of the hydrogen-bonding interaction of C with G in a nonassociating solvent. In contrast, our measured ΔH of C:G base-pair formation in chloroform is ?5.77 ± 0.20 kcal/mol; thus, the absolute value of the enthalpy of hydrogen bonding in the C:G base pair is greater in o-dichlorobenzene than in chloroform. Since chloroform is a solvent known to form hydrogen bonds, the decrease in enthalpic contribution to C:G base pairing in chloroform is due to the formation of hydrogen bonds between the bases and the solvent. The ΔH of hydrogen bonding of G with C reported here differs from previous indirect estimates: Our measurements indicate the ΔH is 50% less in magnitude than the ΔH based on spectroscopic measurements of the extent of interaction. We have also observed that the enthalpy of hydrogen bonding of C with G in chloroform is greater when G is in excess than when C is in excess. This increased heat is due to the formation of C:G_{n > 1} complexes that we have observed using ¹H-nmr. Although C:G₂ structures have previously been observed in triple-stranded polymeric nucleic acids, higher order structures have not been observed between C and G monomers in nonaqueous solvents until now. By using monomers as a model system to investigate hydrogen-bonding interactions in DNA and RNA, we have obtained the following results: A direct measurement of the ΔH of hydrogen bonding in the C:G complex in two nonaqueous solvents, and the first observation of C:G_{n > 1} complexes between monomers. These results reinforce the importance of hydrogen bonding in the stabilization of various nucleic acid secondary and tertiary structures.     

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.     

Crystallographic characterization of the α,γ C12 helix in hybrid peptide sequences

The solid‐state conformations of two αγ hybrid peptides Boc‐[Aib‐γ⁴(R)Ile]₄‐OMe 1 and Boc‐[Aib‐γ⁴(R)Ile]₅‐OMe 2 are described. Peptides 1 and 2 adopt C₁₂‐helical conformations in crystals. The structure of octapeptide 1 is stabilized by six intramolecular 4 → 1 hydrogen bonds, forming 12 atom C₁₂ motifs. The structure of peptide 2 reveals the formation of eight successive C₁₂ hydrogen‐bonded turns. Average backbone dihedral angles for αγ C₁₂ helices are peptide 1 , Aib; φ (°) = ?57.2 ± 0.8, ψ (°) = ?44.5 ± 4.7; γ⁴(R)Ile; φ (°) = ?127.3 ± 7.3, θ₁ (°) = 58.5 ± 12.1, θ₂ (°) = 67.6 ± 10.1, ψ (°) = ?126.2 ± 16.1; peptide 2 , Aib; φ (°) = ?58.8 ± 5.1, ψ (°) = ?40.3 ± 5.5; ψ⁴(R)Ile; φ (°) = ?123.9 ± 2.7, θ₁ (°) = 53.3 θ 4.9, θ ₂ (°) = 61.2 ± 1.6, ψ (°) = ?121.8 ± 5.1. The tendency of γ⁴‐substituted residues to adopt gauche–gauche conformations about the C^α–C^β and C^β–C^γ bonds facilitates helical folding. The αγ C₁₂ helix is a backbone expanded analog of α peptide 3₁₀ helix. The hydrogen bond parameters for α peptide 3₁₀ and α‐helices are compared with those for αγ hybrid C₁₂ helix. Copyright © 2016 European Peptide Society and John Wiley & Sons.     

Nucleic acid-solvent interactions: temperature dependence of the heat of solution of thymine in water and ethanol

The heats of solution of thymine in water and ethanol have been determined calorimetrically as a function of temperature. These data, along with solubility data, have been used to calculate the thermodynamic quantities (ΔG_t, ΔH_t, ΔS_t and ΔC_p,t) associated with the transfer of thymine from ethanol to water. Since ΔS_t = ?2 cal/mole deg and ΔC_p,t = 0, it has been concluded that hydrophobic bonding does not play an important role in the thermocynamic stability of nucleic acids. However, large heat capacities of solution of thymine are observed in both solvents (ΔC°_p2 = 45 ± 4 cal/mole deg). This is explained in terms of temperature variation in the degree of solvent–solute hydrogen bonding. It is our proposal that the components of macromolecules (i.e., nucleic acid bases and amino acids) do not make all possible hydrogen bonds with the solvent in the vicinity of room temperature. Thus the thermodynamic contribution of hydrogen bonding to the stability of macromolecules in aqueous solution must be reassessed.     

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.     

Solvent‐dependent conformation of amylose tris(phenylcarbamate) as deduced from scattering and viscosity data

The z‐average mean‐square radius of gyration 〈S²〉_z, the particle scattering function P(k), the second virial coefficient, and the intrinsic viscosity [η] have been determined for amylose tris(phenylcarbamate) (ATPC) in methyl acetate (MEA) at 25°C, in ethyl acetate (EA) at 33°C, and in 4‐methyl‐2‐pentanone (MIBK) at 25°C by light and small‐angle X‐ray scattering and viscometry as functions of the weight‐average molecular weight in a range from 2 × 10⁴ to 3 × 10⁶. The first two solvents attain the theta state, whereas the last one is a good solvent for the amylose derivative. Analysis of the 〈S²〉_z, P(k), and [η] data based on the wormlike chain yields h (the contour length or helix pitch per repeating unit) = 0.37 ± 0.02 and λ^?1 (the Kuhn segment length) = 15 ± 2 nm in MEA, h = 0.39 ± 0.02 and λ^?1 = 17 ± 2 nm in EA, and h = 0.42 ± 0.02 nm and λ^?1 = 24 ± 2 nm in MIBK. These h values, comparable with the helix pitches (0.37–0.40 nm) per residue of amylose triesters in the crystalline state, are somewhat larger than the previously determined h of 0.33 ± 0.02 nm for ATPC in 1,4‐dioxane and 2‐ethoxyethanol, in which intramolecular hydrogen bonds are formed between the C?O and NH groups of the neighbor repeating units. The slightly extended helices of ATPC in the ketone and ester solvents are most likely due to the replacement of those hydrogen bonds by intermolecular hydrogen bonds between the NH groups of the polymer and the carbonyl groups of the solvent. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 729–736, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Three-stranded helix-coil equilibrium in polyuridylic acid-adenosine complex

Interaction of poly U (polyuridylic acid) and adenosine is studied by following the changes in ultraviolet absorbance in the wavelength region near the isochromic wave-length for the complex formation. The interaction is studied as a function of temperature, concentration of adenosine, and ionic strength, while the concentration of poly U was held constant. It is confirmed that only the three-stranded complex with the stoichiometry 1A to 2U is formed and that it dissociates directly into free poly U and adenosine. No discontinuity of any kind was apparent in the melting curves, and poly U was found to possess no ordered structure above 10°C under the conditions used. The results were, therefore, analyzed in terms of an exact helix–coil equilibrium theory using the mismatching model, i.e., assuming that either completely formed base triplet or completely free unbonded bases only exist, and that the two sections of the polymer chains forming closed loops need not contain the same number of unbonded bases. Self-association of free adenosine was taken into consideration. (Base triplet is analog of base pair for a three-stranded helical complex. It refers to a unit of three coplanar bases, in this case two uracils and one adenine, hydrogen bonded to one another to form a triplet. Such triplets may stack over one another along the helical axis, and when they are so stacked the bases of two triplets next to each other may have stacking interactions between them.) The values for enthalpy and entropy changes, both per mole of base triplets, were obtained for the following processes at neutral pH and moderate to high salt concentrations. (1) Growfh: Binding of one adenosine molecule to two uracil residues (one from each poly U strand) to form a base triplet next to an already formed base triplet with which it has stacking interactions, a process that involves both hydrogen bonding and base stacking interactions, ΔH_s, = ?19 ± 2 kcal, ΔS_s = ?55 ± 6 clausius; (2) Initiation: Binding of one adenosine molecule to two uracil residues (one from each poly U strand) to form an isolated base triplet, a process that involves only hydrogen bonding interactions, ΔH_b* = 4.5 ± 2 kcal, ΔS_b* = 6.6 ± 3 clausius; and (3)Interruption: Unstacking of two stacked base triplets initially next to each other by formation of an interruption (viz. a closed loop) between them, a process that involves only base stacking interactions, ΔH_b = 23.5 ± 3 kcal, ΔS_b = 61.6 ± 7 clausius, where the entropy changes include contributions other than the configurational entropy of closed loops. The discrepancy between our results and the calorimetric ΔH_s of ?13 kcal is attributed to (i) the possible effects of salt arid polymer on the self-association of free adenosine, (ii) the uncertainty in the value of the parameter for the probability of ring closure, and (iii) the contributions due to the partial molal enthalpy of the solvent and the unstacking of any poly U structure to the calorimetric enthalpy.     

Effects of sugars and polyols on the sol-gel transition of k-carrageenan: calorimetric study

The effects of sugars and polyols on the sol-gel transition of k-carrageenan were studied in 0.025 m KCl by means of differential scanning calorimetry. Addition of these compounds invariably raised the gelling temperature T_g, with an increase in their concentration, accompanying a decreased (less negative) enthalpy of gelation, ΔH_g. This indicates that it is not ΔH_g but the entropy of gelatin, ΔS_g, which plays an essential role in the gel stabilizatiion by them, thus differing from the enthalpy-driven stabilization by addition by KCl, ethanol or carrageenan. It seemed that such a large change in ΔS_g relative to ΔH_g predominantly occurs in the process of replacing polymer solvent hydrogen bonds by polymer—polymer hydrogen bonds with minor contribution of the conformational entropy of polymer chains. The gel-stabilizing ability of different sugars and polyols is discussed in terms of their different influences on the structure of water.     

Large contributions of coupled protonation equilibria to the observed enthalpy and heat capacity changes for ssDNA binding to Escherichia coli SSB protein

Many macromolecular interactions, including protein‐nucleic acid interactions, are accompanied by a substantial negative heat capacity change, the molecular origins of which have generated substantial interest. We have shown previously that temperature‐dependent unstacking of the bases within oligo(dA) upon binding to the Escherichia coli SSB tetramer dominates the binding enthalpy, ΔH_obs, and accounts for as much as a half of the observed heat capacity change, ΔC_p. However, there is still a substantial ΔC_p associated with SSB binding to ssDNA, such as oligo(dT), that does not undergo substantial base stacking. In an attempt to determine the origins of this heat capacity change, we have examined by isothermal titration calorimetry (ITC) the equilibrium binding of dT(pT)₃₄ to SSB over a broad pH range (pH 5.0–10.0) at 0.02 M, 0.2 M NaCl and 1 M NaCl (25°C), and as a function of temperature at pH 8.1. A net protonation of the SSB protein occurs upon dT(pT)₃₄ binding over this entire pH range, with contributions from at least three sets of protonation sites (pK_a1 = 5.9–6.6, pK_a2 = 8.2–8.4, and pK_a3 = 10.2–10.3) and these protonation equilibria contribute substantially to the observed ΔH and ΔC_p for the SSB‐dT(pT)₃₄ interaction. The contribution of this coupled protonation (∼ −260 to −320 cal mol⁻¹ K⁻¹) accounts for as much as half of the total ΔC_p. The values of the “intrinsic” ΔC_p,0 range from −210 ± 33 cal mol⁻¹ °K⁻¹ to −237 ± 36 cal mol⁻¹K⁻¹, independent of [NaCl]. These results indicate that the coupling of a temperature‐dependent protonation equilibria to a macromolecular interaction can result in a large negative ΔC_p, and this finding needs to be considered in interpretations of the molecular origins of heat capacity changes associated with ligand‐macromolecular interactions, as well as protein folding. Proteins 2000;Suppl 4:8–22. © 2000 Wiley‐Liss, Inc.     

The triple helix ⇌ coil conversion of collagen-like polytripeptides in aqueous and nonaqueous solvents. Comparison of the thermodynamic parameters and the binding of water to (L-Pro-L-Pro-Gly)n and (L-Pro-L-Hyp-Gly)n

The collagen-like peptides (L -Pro-L -Pro-Gly)_n and (L -Pro-L -Hyp-Gly)_n with n = 5 and 10, were examined in terms of their triple helix ? coil transitions in aqueous and nonaqueous solvents. The peptides were soluble in 1,2-propanediol containing 3% acetic acid and they were found to form triple-helical structures in this solvent system. The water content of the solvent system and the amount of water bound to the peptides were assayed by equilibrating the solvent with molecular sieves and carrying out Karl Fischer titrations on the solvent phase. After the solvent was dehydrated, much less than one molecule of water per tripeptide unit was bound to the peptides. Since the peptides remained in a triple-helical conformation, the results indicated that water was not an essential component of the triple-helical structure. Comparison of peptides with the same chain length demonstrated that the presence of hydroxyproline increased the thermal stability of the triple helix even under anhydrous conditions. The results, therefore, did not support recent hypotheses that hydroxyproline stabilizes the triple helix of collagen and collagen-like peptides by a specific interaction with water molecules. Analysis of the thermal transition curves in several solvent systems showed that although the peptides containing hydroxyproline had t_m values which were 18.6° to 32.7°C higher, the effect of hydroxyproline on ΔG was only 0.1 to 0.3 kcal per tripeptide unit at 25°C. The results suggested, therefore, that the influence of hydroxyproline on helical stability may be explained by intrinsic effects such as dipole–dipole interactions or by changes in the solvation of the peptides by alcohol, acetic acid, and water. A direct calorimetric measurement of the transition enthalpy for (L -Pro-L -Pro-Gly)_n in 3% or 10% acetic acid gave a value of ?1.84 kcal per tripeptide unit for the coil-to-helix transition. From the value for enthalpy and from data on the effects of different chain lengths on the thermal transition, it was calculated that the apparent free energy for nucleation was +5 kcal/mol at 25°C (apparent nucleation parameter = 2 × 10^?4 M^?2). The value was dependent on solvent and on chemical modification of end groups.     

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.     

Possible formation of the left-handed alpha-helix of poly-L-serine

A conformational study of poly-L -serine has shown that it can exist in the left-handed α-helical form. A study of a pair of peptide units with the serine sidegroup attached to the α carbon atom linking the two units showed that O? H ?O hydrogen bonds between the OH group of the side chain and a carbonyl oxygen of the first peptide group in the backbone can occur in two regions of ?, namely, ? = 15°–30° for χ₁ = 300° and for ? = 225°-230° for ? = 60°. The latter is close to a possible left-handed helix of poly-L -serine, stabilized by N? H ?O hydrogen bonds. From a study of contact criteria, the best conformation for this helix is found to be ? = 227°, Ψ = 238°, χ₁ = 65° which has n = 3.65, h = 1.51 A. The N? H ?O hydrogen bond has a length of 2.90 A. (6°) and the O? H ?O hydrogen bond is of length 2.60 A. (0°). There are no other bad short contacts in the structure. The cylindrical coordinates of the atoms, as well as a perspective view of the structure arc given in this paper.     

Unfolding of an α-helix in peptide crystals by solvation: Conformational fragility in a heptapeptide

The structure of the peptide Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-OMe has been determined in crystals obtained from a dimethylsulfoxide–isopropanol mixture. Crystal parameters are as follows: C₃₈H₆₉N₇O₁₀ · H₂O · 2C₃H₇OH, space group P2₁, a = 10.350 (2) Å, b = 26.084 (4) Å, c = 10.395(2) Å, β = 96.87(12), Z = 2, R = 8.7% for 2686 reflections observed > 3.0 σ (F). A single 5 → 1 hydrogen bond is observed at the N-terminus, while two 4 → 1 hydrogen bonds characteristic of a 3₁₀-helix are seen in the central segment. The C-terminus residues, Ala(6) and Leu(7) are expended, while Val(5) is considerably distorted from a helical conformation. Two isopropanol molecules make hydrogen bonds to the C-terminal segment, while a water molecule interacts with the N-terminus. The structure is in contrast to that obtained for the same peptide in crystals from methanol-water [ I. L. Karle, J. L. Flippen-Anderson, K. Uma, and P. Balaram (1990) Proteins: Structure, Function and Genetics, Vol. 7, pp. 62–73] in which two independent molecules reveal an almost perfect α-helix and a helix penetrated by a water molecule. A comparison of the three structures provides a snapshot of the progressive effects of solvation leading to helix unwinding. The fragility of the heptapeptide helix in solution is demonstrated by nmr studies in CDC1₃ and (CD₃)₂SO. A helical conformation is supported in the apolar solvent CDCl₃, whereas almost complete unfolding is observed in the strongly solvating medium (CD₃)₂SO. © 1993 John Wiley & Sons, Inc.     

The folding pathway of glycosomal triosephosphate isomerase: structural insights into equilibrium intermediates

The guanidine hydrochloride‐induced conformational transitions of glycosomal triosephosphate isomerase (TIM) were monitored with functional, spectroscopic, and hydrodynamic measurements. The equilibrium folding pathway was found to include two intermediates (N₂?I₂?2M?2U). According to this model, the conformational stability parameters of TIM are as follows: ΔG_I2‐N2 = 5.5 ± 0.6, ΔG_2M‐I2=19.6 ± 1.6, and ΔG_2U‐2M = 14.7 ± 3.1 kcal mol^?1. The I₂ state is compact (α_SR = 0.8); it is able to bind 8‐anilinonaphthalene‐1‐sulfonic acid ANS and it is composed of ～45% of α‐helix and tertiary structure content compared with the native enzyme; however, it is unable to bind the transition‐state analog 2‐phosphoglycolate. Conversely, the 2M state lacks detectable tertiary contacts, possesses ～10% of the native α‐helical content, is significantly expanded (α_SR = 0.2), and has low affinity for ANS. We studied the effect of mutating cysteine residues on the structure and stability of I₂ and 2M. Three mutants were made: C39A, C126A, and C39A/C126A. The replacement of C39, which is located at β₂, was found to be neutral. The I₂–C126A state, however, was prone to aggregation and exhibited an emission maximum that was 3‐nm red‐shifted compared with the I₂–wild type, indicating solvent exposure of W90 at β₄. Our results suggest that the I₂ state comprises the (βα)_1‐4β₅ module in which the conserved C126 residue located at β₅ defines the boundary of the folded segment. We propose a folding pathway that highlights the remarkable thermodynamic stability of this glycosomal enzyme. Proteins 2012; © 2012 Wiley Periodicals, Inc.     

Conformational transitions of a dipeptide in water: Effects of imposed pathways using umbrella sampling techniques

The free energy difference between two states of a molecular system separated by an energy barrier can generally be computed using the technique of umbrella sampling along a chosen reaction coordinate or pathway. The effect of a particular choice of pathway upon the obtained free energy difference is investigated by molecular dynamics simulation of a model system consisting of a glycine dipeptide in aqueous solution. Two different reaction coordinates connecting the so-called C₅ and C₇ conformations, one involving intramolecular hydrogen bonds and the other involving the peptide ?, ψ angles, are considered. The Gibbs free energy differences ΔG(C₅ – C₇) are small in both cases, 1.5 ± 1 kJ mol^?1 and 2.2 ± 1 kJ mol ^?1, respectively. The two different reaction coordinates yield free energy differences that are identical to within their statistical error. It is found that the exchange of solute–solute, solute–water, and water–water hydrogen bonds involves free energy changes of less than k_BT, which points at the existence of a multitutde of low free energy pathways connecting the C₅ and C₇ dipeptide conformations. © 1994 John Wiley & Sons, Inc.     

Accommodation of a D-Phe residue into a right-handed 310-helix: Structure of Boc-D-Phe-(Aib)4-Gly-L-Leu-(Aib)2-Ome,an analogue of the amino terminal segment of antiamoebins and emerimicins

The crystal structure of the nonapeptide Boc-D -Phe-Aib-Aib-Aib-Aib-Gly-Leu-Aib-AibOMe (I), which is an analogue of the N-terminal sequence of antiamoebins and emerimicins, establishes a completely 3₁₀-helical conformation with seven successive intramolecular 4 → 1 hydrogen bonds. The average, ?, ψ values for residues 1–8 are ?59° and ?32°, respectively. Crystal parameters are C₄₇H₇₇N₉O₁₂, space group P1, a = 10.636(4) Å, b = 11.239(4) Å, c = 12.227(6) Å, α = 101.17(4)°, β = 97.22(4)°, γ = 89.80(3)°, Z = 1, R = 5.95% for 3018 data with |F₀| > 3α(F), resolution 0.93 Å. The use of the torsion angle κ = C(i ? 1)N(i)C^α(i)C^β(i), where κ = 68° for D -Phe and κ = 164° for L -Leu, confirms the opposite configurations of these residues. The ?, ψ values of ?62° and ?32° at D -Phe are unusual, since this region is characteristic of residues with L configurations. Peptide I possesses only two chiral residues of opposing configuration. The observed right-handed 3₁₀-helical structure suggests that helix sense has probably been determined by the stereo-chemical preferences of the Leu residue. © 1993 John Wiley & Sons, Inc.     

Conformational studies on copolymers of L-lysine and L-leucine: circular dichroism and potentiometric titration studies

Conformational aspects of a series of copolymers of L -Leucine and L -leucine [poly-(Lys^xLeu^y)] containing 0 to 0.41 mole fraction L -leucine have been studied by circular dichroism (CD) and potentiometric titration in 0.05M KF solution. CD studies on the α-helical conformation showed a dependence of the magnitude of the CD ellipticity band at 222 nm on copolymer composition; the [θ]₂₂₂ decreasing with higher leucine contents. This was interpreted as the result of an increase of the hydrophobicity of the environment of the amide group due to the presence of the leucyl residues. Values of the Zimm-Rice parameter, σ, for the copolymers were obtained from the potentiometric titrations and used to fit theoretical curves to the experimental data. Using the variation of σ with polymer composition, a value of σ for the leucyl residue was estimated to be 6.3 × 10^?2, assuming independence of σ on the amino acid sequence in the copolymer. The free energy change for the conversion of one mole residue from uncharged helix to uncharged coil, ΔG_hc°, was also obtained from the titration data for each copolymer up to a leucine mole fraction of 0.16; a value of 385 cal mole^?1 was estimated for ΔG_hc° for a leucyl residue. These values for σ and ΔG_hc° are compared with other values in the literature for various amino acid residues obtained from titration and melting curve data.     

The role of water in the crystal structure of N-acetyl-L-4-hydroxyproline

The crystal structure of N-acetyl-L -4-hydroxyproline (Hyp) was determined by direct methods. (The crystal is orthorhombic with the space group P2₁2₁2₁.) The acetyl group is in the trans conformation and the pyrrolidine ring puckers at C^γ (C^sC^γ envelope), as in most Hyp residues. According to the rotation angle ψ = ?30°, the N-acetyl-L -4Hyp has the same conformation as an α-helix of prolyl residues. The crystal packing is stabilized by hydrogen bonds between three different molecules and the same molecule of water. One of the water bridges involves the carbonyl of the N-acetyl group of one molecule and the hydrogen atom of the 4-OH group of another. Such an arrangement has been proposed to explain the high stability of (Gly-L -Pro-L -4Hyp)_n. A second bridge involves the two hydrogens of the water molecule and the carbonyl groups of two neighbouring molecules, as already proposed in a dihydrated model of collagen. These experimental features, which are discussed in relation to the different models of collagen, allow us to propose an hypothetical arrangement for the water molecule which is strongly retained in the triple helix of (Gly-L -Pro-L -4Hyp)_n.     

Thermodynamics of oligo (A) N -2poly(U) from the dependence of the temperature of the helix-coil transition on oligomer concentration

On the basis of elementary two-state, ideal solution thermocynamics, a modified expression for the melting of oligo. polynucleotide helices is derived which is applicable to variations in T_m^N and/or oligomer concentration, C_m with oligomer length, N: ((I)) ΔH_r is the enthalpy per helix residue, i.e., per base-pair or base-triplet, V_r^f is the thermodynamic “available” or “reaction” volume, in liters/mole of helical residues; and n is the number of polynucleotide strands, e.g., n = 2 for oligo (A)_N·2 poly(U)∞. Some earlier treatments have engendered confusion in the interpretation of the “reaction volume,” but with the derivation herein, the entropic origin and physical significance of V_r^f is unequivocal. The following approximation was arrived at for the reduction expected in the configurational entropy, ΔS_r^{conf, ∞}, for (A)∞·2(U)∞, when the poly(A), strand is substituted for by an equivalent strand of contiguous oligo(A)_N,′s: ((II)) This adjustment of ΔS_r^{conf, ∞} represents the source of the coefficient to 1/T_m^∞ in expression (I). The expectation that ΔS_r^{conf, N} < ΔS_r^{conf, ∞} is due to the effect of releasing normal internucleotide configurational restrictions every Nth residue in one-third of the strands of the (A)_N·2(U)∞ helix. Although the reduction in ΔS_r^{conf, ∞} (II) may seem small (i.e., only 5.5% for the tetramer), its effect on the magnitude of V_r^f in expression (I) is exponential. Thus, without these considerations the quantitative applicability of earlier expressions is questionable. By examining the variation in T_m^N with c_m for a single N, all assumptions, required for evaluating V_r^f or the entropic effects of discontinuities in the (A)_N strand are avoided in the determination of a reliable enthalpy. We have therefore examined the system ((III)) and obtained a ΔH_r = 12.58 ± 0.08 kcal per mole (A)·2(U) base-triplets between 5 and 2.5°C. That this value for ΔH_r is in such excellent agreement with all calorimetric values reported for (A)∞·2(U)∞ suggests that the enthalpy for reaction(III) is not significantly affected by disconnections in the backbone of (A)₄·2(U)∞. From (I), V_r^f = 6.0 × 10^?4 1/mole or 1 Å ³per helical residue. ΔH_r°, corrected for residual single-strand stacking in (A)₄, is in excellent agreement with that found earlier for (A)₁·2(U)∞. A residual heat capacity of 90 kcal(±20) per mole (A)·2(U) base-triplets per °C is deduced from the decrease of ΔH_r° with temperature.     

Thermodynamic characterization of ethidium bromide binding to a unique site on yeast tRNAphe.

A self-consistent thermodynamic characterization of the binding of ethidium to yeast phenylalanine-specific tRNA at 25°C, pH 7.0, in 11 nM MgCl₂, 375 nM NaCl, and 25 mM sodium phosphate has been obtained. Two ethidium molecules bind per tRNA under these conditions. The stronger site has a dissociation constant equal to 1.9 ± 0.5 μM and ΔH_dis°′ = 12 ± 1 Kcal/mol, and the weaker sites has a dissociation constant equal to 24 ± 9 μM and ΔH_dis°′ = 8.9 ± 1.5 Kcal/mol. The average calorimetric ΔH_dis°′ for the to sites 10.6 ± 0.4 kcal/mol. The thermodynamics of binding to the stranger sites are most probably the thermodynamics of interaction between A·U (6) and A·U (7), the unique site identified by Jones and Kearns. The binding is enthalpically driven and classical hydrophobic interactions do not appear to be important in the binding reaction.