Kinetics of the helix/coil transition of the collagen-like peptide (Pro-Hyp-Gly)10

This article measures the rates of folding and unfolding of the collagen-like peptide (Pro-Hyp-Gly)(10) over overlapping concentration and temperature ranges. The data allow calculation of the orders of the folding and the unfolding reactions, the effective Arrhenius activation energies, and numerical solution of the differential equation controlling the helix/coil transition during temperature scanning. The resulting predictions of helicity closely followed DSC measurements of the peptide in both up- and down-scanning modes, confirming the validity of the theoretical equations governing the kinetics of the folding/unfolding process. In both up- and down-scanning, three regions were apparent: "quasistatic," "rate," and "mixed." At very low scanning rates, a quasistatic region revealed a broad, short endotherm that was independent of scanning rate, but dependent on concentration and equal to the equilibrium endotherm. At high up-scanning rates, the "rate region" endotherm was sharp and tall and T(max) increased with scanning rate. In down-scanning, the "rate peak" was very broad and very short and T(max) decreased with scanning rate. The "mixed region" showed nascent "rate" and nascent "quasistatic" peaks, which were evident in the same up-scan under certain conditions. Comparison of (Pro-Hyp-Gly)(10) and (Pro-Pro-Gly)(10) showed that the higher temperature stability of (Pro-Hyp-Gly)(10) is due mainly to its slower rate of unfolding and higher activation energy.     

Helix–coil stability constants for amino acids in nonaqueous solvents. Studies of random poly(γ-benzyl-L-glutamate-co-L-alanine) and poly(γ-benzyl-L-glutamate-co-L-leucine) in a mixed solvent

The host–guest technique has been applied to the determination of the helix–coil stability constants of two naturally occurring amino acids, L -alanine and L -leucine, in a nonaqueous solvent system. Random copolymers containing L -alanine and L -leucine, respectively, as guest residues and γ-benzyl-L -glutamate as the host residue were synthesized. The polymers were fractionated and characterized for their amino acid content, molecular weight, and helix–coil transition behavior in a dichloroacetic acid (DCA)–1,2-dichloroethane (DCE) mixture. Two types of helix–coil transitions were carried out on the copolymers: solvent-induced transitions in DCA–DCE mixtures at 25°C and thermally induced transitions in a 82:18 (wt %) DCA–DCE mixture. The thermally induced transitions were analyzed by statistical mechanical methods to determine the Zimm-Bragg parameters, σ and s, of the guest residues. The experimental data indicate that, in the nonaqueous solvent, the L -alanine residue stabilizes the α-helical conformation more than the L -leucine residue does. This is in contrast to their behavior in aqueous solution, where the reverse is true. The implications of this finding for the analysis of helical structures in globular proteins are discussed.     

Two types of tripeptide conformation in collagen. Calculation of the structure of (Gly-Pro-Ser)n and (Gly-Val-Hyp)n polytripeptides

Conformational analysis of polypeptides (Gly-Pro-Ser)n and (Gly-Val-Hyp)n was carried out for collagen-like triple helical complexes (coiled coils with screw symmetry). The lowest energy structure of the first polymer (helical parameters t 52,8, h 0,282 nm) is very close to that of (Gly-Pro-Hyp)n. The hydroxyl group of a serine residue does not form any intramolecular hydrogen bonds in this structure. (Gly-Val-Hyp)n triple complex is shown to unwind to t 7,7, h 0,297 nm as a result of optimization procedure. These findings confirm the assumption, made earlier on the basis of conformational analysis of (Gly-Pro-Hyp)n, (Gly-Pro-Ala)n, (Gly-Ala-Hyp)n, (Gly-Ala-Ala)n, that the collagen triple helix contains stable wound triplets with proline in the second position, while the absence of imino acid in the 2nd position facilitates the unwinding of the triple helix. Thus, a collagen helix appears to have different parameters for the sites differing in the amino acid sequence. The values measured in the X-ray experiments (h 0,29 nm, t' 36) should be considered as a result of averaging. The model allows to reconcile the X-ray data for collagen and crystalline (Gly-Pro-Pro)10 oligomer.     

Conformational analysis of polytripeptides (Gly-Pro-Ala)n, (Gly-Ala-Hyp)n,and (Gly-Ala-Ala)n in connection with the problem of collagen structure

Conformational analysis of triple helics of a type of collagen was performed with typical collagen tripeptide sequences based on Gly-Pro-Ala, Gly-Ala-Hyp, and Gly-Ala-Ala. During energy minimization, the possibility of continual deformation of the pyrrolidine cycle was taken into account in order to achieve better accuracy in the resulting structure. The (Gly-Pro-Ala)_n structure is almost isomorphic to the (Gly-Pro-Hyp)_n structure obtained in the previous work [Tumanyan, V. G. & Esipova, N.G. (1982) Biopolymers 21 , 475–497]. For a collagen-type structure, the optimal conformation of (Gly-Ala-Hyp)_n tends to have a decreased unit twist (t = 15°), although the energy advantage with respect to the conformation with t = 45° is not so significant. A similar situation is observed for (Gly-Ala-Ala)_n. In this case, the energy decrease during unwinding to t = 15° from t = 45° is quite small. The conformations of (Gly-Ala-Hyp)_n and (Gly-Ala-Ala)_n with t = 15° exhibit a similarity with a triple complex of polyproline II helices—a noncoiled coil such as (Gly-Pro-Hyp)_n and (Gly-Pro-Ala)_n. A similar structure may be postulated for subcomponent cq1 of the first component of a human complement containing substantial Gly-X-Pro and Gly-X-Y tripeptide derivatives in the primary structure (X, Y = any amino acid). The results suggest that the observed helical symmetry of collagen (t = 36°) is a consequence of superposition of diffraction patterns (for sufficiently long segments) from various helices (t varies from ～15° for Gly-X-Hyp and Gly-X-Y to ～56° for Gly-Pro-Ala). For short alternating segments, some unification of different helical structures is possible.     

Proton magnetic resonance of the triple helix of poly(prolylprolylglycyl)n with defined degree of polymerization

Proton magnetic resonance spectra at 220 MHz were obtained for deuterium oxide and aqueous solutions of the polytripeptides (Pro-Pro-Gly)_n, which were synthesized as collagen models by the modified solid phase method. At higher temperatures, the signals of the proline C_a-protons for the peptides with n ≦ 5 and for those with n = 10 and 15 demonstrate the presence of cis and trans isomers with respect to the Gly-Pro or Pro-Pro C-N bonds. Glycine C_a-protons give typical AB type patterns. At lower temperatures, as the peptides with n = 5, 10 and 15 form triple helices, all of the resonance peaks become broad, but the whole form of the spectrum is quite similar to that of poly(l-proline) form II. The glycine C_a-proton resonances become barely detectable and the upfield peak of the two proline C_a-proton resonances fade away. At the same time, a new glycine NH resonance appears at a field slightly higher than that of a random coil. It seems to suggest that the formation of triple helices accompanies the conversion of cis proline peptide bonds into all trans bonds, and that the glycine residue environment completely changes in the helix.     

Solvent- and salt-induced coil–helix transition of alkali metal salts of poly(L-glutamic acid) in aqueous organic solvents

The coil–helix transition has been studied for alkali metal salts of poly (L -glutamic acid) (PLG), i.e., PLGLi, -Na, -K, and -Cs, in aqueous organic solvent systems. Dependence of the transition on the solvent composition has been qualitatively discussed in terms of the solvent dielectric constant D, Gutmann's acceptor number AN, and water activity a_w. The helix formation induced by addition of alkali chlorides has also been studied. The sharpness of the transition has been interpreted as a measure of reduction of electrostatic energy of helical PLG through contact ion-pair formation between a counterion and carboxyl anion.     

The formation of the triple helix in a mixed solution of (Pro-Pro-Gly)n and (Pro-Pro-Gly)n'. A model of molecular size recognition

A theoretical analysis is given of the triple-helix–random-coil transition in a mixed solution of poly(Pro-Pro-Gly)_n with two different but defined degrees of polymerization n and n′. Because of the highly cooperative nature of this helix–coil transition, each polypeptide chain tends to form a triple helix with other polypeptide chains with the same degree of polymerization (size recognition). Occurrence of triple helices consisting of polypeptide chains with different degrees of polymerization (error in recognition) is studied in detail as a function of the cooperativity, and n and n′. Implication of this analysis for molecular recognition in general is discussed.     

Structure of d(T)n.d(A)n.d(T)n: the DNA triple helix has B-form geometry with C2'-endo sugar pucker.

The polynucleotide helix d(T)n.d(A)n.d(T)n is the only deoxypolynucleotide triple helix for which a structure has been published, and it is generally assumed as the structural basis for studies of DNA triplexes. The helix has been assigned to an A-form conformation with C3'-endo sugar pucker by Arnott and Selsing [1974; cf. Arnott et al. (1976)]. We show here by infrared spectroscopy in D2O solution that the helix is instead B-form and that the sugar pucker is in the C2'-endo region. Distamycin A, which binds only to B-form and not to A-form helices, binds to the triple helix without displacement of the third strand, as demonstrated by CD spectroscopy and gel electrophoresis. Molecular modeling shows that a stereochemically satisfactory structure can be build using C2'-endo sugars and a displacement of the Watson-Crick base-pair center from the helix axis of 2.5 A. Helical constraints of rise per residue (h = 3.26 A) and residues per turn (n = 12) were taken from fiber diffraction experiments of Arnott and Selsing (1974). The conformational torsion angles are in the standard B-form range, and there are no short contacts. In contrast, we were unable to construct a stereochemically allowed model with A-form geometry and C3'-endo sugars. Arnott et al. (1976) observed that their model had short contacts (e.g., 2.3 A between the phosphate-dependent oxygen on the A strand and O2 in the Hoogsteen-paired thymine strand) which are generally known to be outside the allowed range.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetic and thermodynamic parameters for tRNA binding to the ribosome and for the translocation reaction.

Kinetic analyses of tRNA binding to the ribosome and of the translocation reaction showed the following results. 1) The activation energy for the P site binding of AcPhe-tRNA to poly(U)-programmed ribosomes is relatively high (Ea = 72 kJ mol-1; 15 mM Mg2+). If only the P site is occupied with deacylated tRNA(Phe), then the E site can be filled more easily with tRNA(Phe) (no activation energy measurable) than the A site with AcPhe-tRNA (Ea = 47 kJ mol-1; 15 mM Mg2+). 2) A ribosome with blocked P and E sites represents a standard state of the elongation cycle, in contrast to a ribosome with only a filled P site. The two states differ in that AcPhe-tRNA binding to the A site of a ribosome with prefilled P and E sites requires much higher activation energy (87 versus 47 kJ mol-1). The latter reaction simulates the allosteric transition from the post- to the pretranslocational state, whereby the tRNA(Phe) is released from the E site upon occupation of the A site (Rheinberger, H.-J., and Nierhaus, K. H. (1986) J. Biol. Chem. 261, 9133-9139). The reversed transition from the pre- to the posttranslocational state (translocation reaction) requires about the same activation energy (90 kJ mol-1). 3) Both elongation factors EF-Tu and EF-G drastically reduce the respective activation energies. 4) The rate of the A site occupation is slower than the rate of translocation in the presence of the respective elongation factors. The data suggest that the A site occupation rather than, as generally assumed, the translocation reaction is the rate-limiting step of the elongation cycle.     

Helix coil transitions of d (A)n·d(T)n,d(A-T)n·d(A-T)n,and d(A-A-T)n·d(A-T-T)n; evaluation of parameters governing DNA stability

The thermally induced helix-coil transitions of three A-T DNAs, d(A)_n·d(T)_n, d(A-T)_n·d(A-T)_n, and d(A-A-T)_n·d(A-T-T)_n, were studied. Experimental transition curves of the DNAs were analyzed using the loop entropy model of DNA melting. The calculation of the melting curve of d(A-A-T)_n·d(A-T-T)_n is presented using the integral equation formalism of Goel and Montroll. The aim of this work was to evaluate thermodynamic parameters which govern DNA stability and to test the theoretical model employed in the analysis. Our results show (1) an excellent over-all agreement between theory and experiment, (2) a loop entropy exponent k = 1.55 ± 0.05 provided the best fit to all the polymer transition curves, (3) the evaluated stacking free energies reflect the relative stability of the DNAs, and (4) the stacking energies of the ApA·TpT dimer evaluated from d(A)_n·d(T)_n and d(A-A-T)_n·d(A-T-T)_n differ. The last result is consistent with different conformations for the dimer in these two polymers.     

Differential stability of the triple helix of (Pro-Pro-Gly)10 in H2O and D2O: thermodynamic and structural explanations

(Pro-Pro-Gly)10 [(PPG10)], a collagen-like polypeptide, forms a triple-helical, polyproline-II structure in aqueous solution at temperatures somewhat lower than physiological, with a melting temperature of 24.5 degrees C. In this article, we present circular dichroism spectra that demonstrate an increase of the melting temperature with the addition of increasing amounts of D2O to an H2O solution of (PPG)10, with the melting temperature reaching 40 degrees C in pure D2O. A thermodynamic analysis of the data demonstrates that this result is due to an increasing enthalpy of unfolding in D2O vs. H2O. To provide a theoretical explanation for this result, we have used a model for hydration of (PPG)10 that we developed previously, in which inter-chain water bridges are formed between sterically crowded waters and peptide bond carbonyls. Energy minimizations were performed upon this model using hydrogen bond parameters for water, and altered hydrogen bond parameters that reproduced the differences in carbonyl oxygen-water oxygen distances found in small-molecule crystal structures containing oxygen-oxygen hydrogen bonds between organic molecules and H2O or D2O. It was found that using hydrogen bond parameters that reproduced the distance typical of hydrogen bonds to D2O resulted in a significant lowering of the potential energy of hydrated (PPG)10. This lowering of the energy involved energetic terms that were only indirectly related to the altered hydrogen bond parameters, and were therefore not artifactual; the intra-(PPG10) energy, plus the water-(PPG10) van der Waals energy (not including hydrogen bond interactions), were lowered enough to qualitatively account for the lower enthalpy of the triple-helical conformation, relative to the unfolded state, in D2O vs. H2O. This result indicates that the geometry of the carbonyl-D2O hydrogen bonds allows formation of good hydrogen bonds without making as much of an energetic sacrifice from other factors as in the case of hydration by H2O.     

The crystal structure of a collagen-like polypeptide with 3(S)-hydroxyproline residues in the Xaa position forms a standard 7/2 collagen triple helix

Collagen has a triple helical structure comprising strands with a repeating Xaa-Yaa-Gly sequence. L-Proline (Pro) and 4(R)-hydroxyl-L-proline (4(R)Hyp) residues are found most frequently in the Xaa and Yaa positions. However, in natural collagen, 3(S)-hydroxyl-L-proline (3(S)Hyp) occurs in the Xaa positions to varying extents and is most common in collagen types IV and V. Although 4(R)Hyp residues in the Yaa positions have been shown to be critical for the formation of a stable triple helix, the role of 3(S)Hyp residues in the Xaa position is not well understood. Indeed, recent studies have demonstrated that the presence of 3(S)Hyp in the Xaa positions of collagen-like peptides actually has a destabilizing effect relative to peptides with Pro in these locations. Whether this destabilization is reflected in a local unfolding or in other structural alterations of the collagen triple helix is unknown. Thus, to determine what effect the presence of 3(S)Hyp residues in the Xaa positions has on the overall conformation of the collagen triple helix, we determined the crystal structure of the polypeptide H-(Gly-Pro-4(R)Hyp)3-(Gly-3(S)Hyp-4(R)Hyp)2-(Gly-Pro-4(R)Hyp)4-OH to 1.80 A resolution. The structure shows that, despite the presence of the 3(S)Hyp residues, the peptide still adopts a typical 7/2 superhelical symmetry similar to that observed in other collagen structures. The puckering of the Xaa position 3(S)Hyp residues, which are all down (Cgamma-endo), and the varphi/psi dihedral angles of the Xaa 3(S)Hyp residues are also similar to those of typical collagen Pro Xaa residues. Thus, the presence of 3(S)Hyp in the Xaa positions does not lead to large structural alterations in the collagen triple helix.     

NMR studies on thermal stability of α‐helix conformation of melittin in pure ethanol and ethanol–water mixture solvents

Thermal stability of the α‐helix conformation of melittin in pure ethanol and ethanol–water mixture solvents has been investigated by using NMR spectroscopy. With increase in water concentration of the mixture solvents (from 0 wt% to ~71.5 wt%) as well as temperature (from room temperature to 60 °C), the intramolecular hydrogen bonds formed in melittin are destabilized and the α‐helix is partially uncoiled. Further, the hydrogen bonds are found to be more thermally stable in pure ethanol than in pure methanol, suggesting that their stability is enhanced with increase in the size of the alkyl groups of alcohol molecules. Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Cooperative binding of 2,2′‐bipyridine into polynucleotide poly(A)–poly(U‐) in an alkaline aqueous solution

UV absorption data analysis has been used to evaluate equilibrium constants of the pH‐induced interaction of 2,2′‐Bipy with polyadenylnic‐polyuridylic acid in aqueous solution. The conditional probabilities hard model has been adopted in treatment of concentration diagrams calculated by the soft modelling‐based Multivariate Curve Resolution‐Alternating Least Squares approach. Intrinsic binding constant (lgK_g = 1.93), and the cooperativity parameter (ω = 340), were calculated as the best fit. The plot of the experimental binding constant versus 2,2′‐Bipy equilibrium concentration shows two modes of ligand with polymer interactions. The equilibrium hard model correctly reproduced the binding constant variations observed in the experiment. The results indicated that ligand binding in two steps is governed by a cooperative process, that is, the enhancement of deprotonated structure stability. It would appear that proposed calculation approach can be used in future combined hard modelling theoretical and soft modelling experimental works. © 2013 Wiley Periodicals, Inc. Biopolymers 99:621–627, 2013.     

Effect of pressure on the helix and random coil forms of poly(D-glutamic acid)

The absorption spectrum of the aqueous solution of acridine orange (AO)-poly–(D -glutamic acid) (PDGA) complex at pH 4.5 (helix form) did not show any wavelength shift, but at pH 7.5 (coil form) changed to the absorption curve of the helix form by compression up to 4500 atm. The ionization degree of PDGA estimated from the electric conductivity of the aqueous solution of PDGA at 4500 atm. was a value of about 5.3%. The entropy of the helix formation of PDGA from the titration data at 1 atm. and 30°C. was negative ?2.98 e.u. It will be concluded in this report that the volume change for coil to helix could be positive for PDGA and negative for AO–PDGA complexes.     

The cytokine stimulating activity of (1-->3)-beta-D-glucans is dependent on the triple helix conformation

The immunomodulating properties of comb-like branched (1-->3)-beta-D-glucans scleroglucan, schizophyllan and lentinan depend on branching pattern, molecular weight and higher-order structure. The effect of weight average molecular weight Mw and higher order structure of scleroglucan, on stimulation of human monocytes cultured in vitro to secrete tumor necrosis factor-alpha (TNF-alpha) was investigated. The higher order structures of the scleroglucan samples were determined by electron microscopy. The data showed that the samples with a linear wormlike, triple helical structure with Mw less than 50 x 10(4) g/mol or larger than 110 x 10(4) g/mol stimulated the monocytes more efficiently than samples with Mw in the range (67-110) x 10(4) g/mol. The denaturation of the linear triple helices by NaOH (> 0.25 M), followed by neutralization yielded blends of linear and macrocyclic topologies with concomitant irreversible reduction of the cytokine inducing activity compared with the untreated scleroglucans. The dose-dependent ability to activate monocytes to cytokine production was not restored following annealing of the denatured-renatured samples, despite the fact that electron micrographs revealed similar structures of these annealed samples to the starting material. Pre-incubation of monocytes with antibodies against cluster of differentiation antigens CD14 or CD11b reduced the scleroglucan potency to stimulate TNF-alpha secretion mainly for mAb against CD14 in the presence of serum.     

Kinetic analysis of the annealing period in the formation of the poly(A)·2poly(U) triple helix

The slow kinetics of annealing processes in multistranded nucleic acids is spectrophotometrically investigated using poly(A)·2poly(U) as a model system. The absorbance changes at specific wavelengths show that double-helical (A·U) base pairs appear as transient intermediates. The annealing process is identified by the enlargement of triple-helical sequences at the cost of (A·U) base pairs and unpaired (U) residues. A large time range in the reorganization of mismatched chain configurations is characterized by a logarithmic dependence on time. This observation is quantitatively described by a kinetic model developed by Jackson. In Jackson's model the rate-limiting process in the slow annealing phase of maximizing triple-helical sequences, is the removal of strand entanglements, knots, and hairpin loops by complete unwinding of those helical stretches which stabilize the mismatched configurations. The results of the present study are briefly discussed in terms of optimum conditions for hybridization experiments and for the preparation of polynucleotide complexes commonly used to produce interferons.