Conformational change of the triple-helical structure. III. Stabilizing forces in the triple helix

The analysis of thermal melting curves of (PPG)_n (n = 10, 12, 14, and 15) and (PPG)_n(APG)_m (PPG)_n (2n + m = 15; m = 1, 3, and 5) revealed that the enthalpy and entropy changes accompanying the transition from the random coil to the triple helix are ?2500 cal and ?6.3 e.u. per one mole of the tripeptide of the form of Pro-Pro-Gly, and ?3100 cal and ?11.2 e.u. per one mole of the tripeptide of the form of Ala-Pro-Gly. The thermal instability of the triple helix composed of Ala-Pro-Gly sequences, compared to the helix of Pro-Pro-Gly sequences, is due to the larger entropy change of Ala-Pro-Gly (?11.2 e.u.) compared to that of Pro-Pro-Gly (?6.3 e.u.), not from the difference in the enthalpy change. The difference in the enthalpy change between Pro-Pro-Gly and Ala-Pro-Gly arises from the hydrophobic bond between two pyrrolidine rings of proline residues formed in the triple helix. Since the enthalpy change for the formation of hydrophobic bonds is positive, it is also concluded that only one hydrogen bond is formed in a tripeptide unit, regardless of the amino acid sequence. The enthalpy change for the formation of this hydrogen bond is ?3100 cal/mol, and that of the hydrophobic bond between two pyrrolidine rings is +600 cal/mol.     

Conformational change of the triple-helical structure. II. Conformation of (Pro-Pro-Gly)n and (Pro-Pro-Gly)n (Ala-Pro-Gly)m(Pro-Pro-Pro-Gly)n in an aqueous solution

Measurements of the molecular weight of (Pro-Pro-Gly)_n and (Pro-Pro-Gly)_n(Ala-Pro-Gly)_m(Pro-Pro-Gly)_n, which were synthesized by the solid-phase method, revealed that they formed a trimer in an aqueous solution, and dissociated into single-stranded chains on warming. Accompanying the transition, a large decrease of optical rotation was observed, like the collagen–gelatin transition. The shape of the trimeric molecule was rodlike, and the dimensions were 12 Å in diameter and 2.8 Å per residue in length, regardless of the length of Ala-Pro-Gly sequences in a peptide chain. The data indicate that both Pro-Pro-Gly sequences and Ala-Pro-Gly sequences from the triple-helical structure similar to that of collagen in aqueous solution. All optical rotational dispersion (ORD) curves of solutions of the peptides were represented by a single-term Drude equation, and the Drude constant λ_c was 200 nm for all peptides regardless of the length of Ala-Pro-Gly sequences. The resemblance between the helical structure formed by Pro-Pro-Gly sequences and that by Ala-Pro-Gly sequences was also suggested by the formation of the hybrid triple helix from two kinds of peptide chains with different lengths of Ala-Pro-Gly sequences.     

Physicochemical analysis of (Pro-Pro-Gly)n with defined molecular weight--temperature dependence of molecular weight in aqueous solution

Sequenced polytripeptides, (Pro-Pro-Gly)_n, (n = 10, 15, 20), with defined molecular weights were synthesized by the solid-phase method. Conformational changes of these sample as a function of temperature were studied by measurements of optical rotation and sedimentation equilibrium. The temperature dependence of optical rotation was shown similar to thermal transition of collagen molecule. Each of these polymers existed as a timer at lower temperature. (Pro-Pro-Gly)₁₀ existed as a monomer at higher temperature, and the others were expected to behave analogously.     

The association reaction of collagen model polypeptides (Pro-Pro-Gly)n

The characterization of recently synthesized (Pro-Pro-Gly)_n, n = 7, 8 is described, along with melting profile studies of its association equilibrium, and thermal quenching studies of the kinetics of its association reaction. The order of the kinetic reaction is about 3, implying that three peptide chains are involved in the activated state of the rate-limiting step. The reaction rate was found to exhibit a negative temperature coefficient. With the (Pro-Pro-Gly)₇ peptide, the concentration dependence of the (Pro-Pro-Gly)_n association equilibrium was observed for the first time. Detailed thermodynamic analysis for these n = 7, 8 data, together with literature data for n = 10, 15, 20 were carried out for both the simple “all-or-none” binding model and for a series of complex equilibrium models. For the latter, all of the (Pro-Pro-Gly)_n data (in 10% acetic acid) are fit best with a maximally cooperative near-neighbor model with a standard enthalpy change ΔH = ?650 cal/mole of residues, and a standard entropy change ΔS = ?14.63 ?10/n cal/deg-mole of residues, wherein the ?10 eu represents an end-effect contribution to the binding free energy. With regard to optical rotatory properties and thermodynamic parameters, the data for the new n = 7, 8 peptides match rather well with the literature data for the n = 10, 15, and 20 peptides. The enthalpic stabilization per residue of the triple-helical form of (Pro-Pro-Gly)_n was nearly an order of magnitude smaller than the enthalpic stabilization per additional proline obtained from direct calorimetric measurements on native collagens of different (and much lower) proline contents by Privalov and Tiktopulo. [Biopolymers (1970) 9 , 127–139.] Possible explanations for this phenomenon are discussed.     

Investigation of the formation of triple helices between (pro-pro-gly)n's of different degrees of polymerization using gel filtration

The properties of triple-helix formation of (Pro-Pro-Gly)_n were studied. The probability of hybridization between the polytripeptides (Pro-Pro-Gly)_n, of different degrees of polymerization was investigated by gel filtrations of mixed solutions of them. Using samples selectively labeled with radioactivity, the elution pattern was detected by uv absorption and liquid scintillation counting. The hybridization was found only when the difference of polymerization is small. The amount of heterohelices (triple helices of the polypeptide chains with different degrees of polymerization) measured by integrated intensity of elution pattern was comparable to the theoretical value of Suezaki and Go. Concentration dependence of the helix–coil transition temperature of (Pro-Pro-Gly)_n was also measured by the use of optical rotation and discussed with the theoretically expected values.     

The enzymic hydroxylation of protocollagen models

1. Synthetic polymers of l-prolyl-l-prolylglycine of defined chain length, (Pro-Pro-Gly)_n, were found to be substrates for the enzyme protocollagen–proline hydroxylase, with optimum chain length n=5. Boiling the polymer (Pro-Pro-Gly)₁₅ increased its activity as a substrate but had no effect on (Pro-Pro-Gly)₅. 2. Protection of both or one of the N- and C-terminal groups made (Pro-Pro-Gly)₃ a better substrate, and collagenase digestion of hydroxylated tert.-pentyloxy-carbonyl-(Pro-Pro-Gly)₃ benzyl ester indicated that the central prolyl residues were the major points of hydroxylation. 3. The results suggest that the long-chain peptides are optimum substrates but that a triple-stranded structure is inhibitory for hydroxylation.     

Note on an inequality for the coefficients in legendre polynomial expansions and its application to the theory of liquid phase transitions

It is shown that if themth derivative of a function is positive, and it has a Legendre polynomial expansion with coefficients,A _n, then (A _m)/(2m+1)≧(A _n)/(2n+1) forn>m. This result is applied to the theory of liquid phase transitions.     

Dynamic light-scattering study of synthetic myosin filaments

Synthetic myosin filaments of rabbit were prepared. Electron microscopy showed that the number-average length (L_n = 470 nm) and sharpness in length distribution (L_w/L_n = 1.03₆) were independent of ionic strengths of 134, 74, and 44 mM, whereas the number ratio of M-filaments (about 15 nm in diameter at the bare zone) to m-filaments (about 10 nm) strongly depended on ionic strength (IS); the major filaments were M-filaments at IS = 134 mM, m-filaments at IS = 74 mM, and almost exclusively m-filaments at IS = 44 mM. Dynamic light scattering showed that the change in diameter with the change in ionic strength by 2-h dialysis was reversible. Combination of dynamic light scattering and sedimentation studies suggested a dynamic equilibrium between M- and m-filaments. Dynamic light-scattering spectra at IS = 134 and 74 mM could be analyzed by a theory for rigid rods, whereas those at IS = 44 mM only by introducing semiflexibility of filaments; m-filaments are more flexible at IS = 44 than at 74 mM.     

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.     

Linear oligopeptides. 56. Critical size for development of β-and α-helical structures of monodispersed homo-l-oligomethionines in the solid state

The infrared absorption properties of monodispersed, chemically and optically pure ‘amino-PEG’-bound linear homooligopeptides having the general formula t-Bx(l-Met)_n NHPEG, n = 1–15 and H₂⁺(l-Met)_nNHPEG, n = 1–14 have been investigated in the solid state. The critical sizes for development of the β-structure and α-helix (n = 3 and n = 13, respectively), and the effect, on the latter, of the solvent from which the solid samples are cast, have been established.     

Polypeptide models of collagen: Properties of (Pro-Pro-betaAla)n.

We have synthesized (Pro-Pro-βAla)_n as a model for collagen. The synthetic polytripeptide, mol wt 6500, exhibits a large negative optical rotation with a very strong negative Cotton effect centered at 216 nm. The optical rotatory dispersion of (Pro-Pro-βAla)_n followed a single-term Drude equation and the λ_c was 195 nm. The rotation decreased markedly on heating with the midpoint of the broad transition at 55°C. Preliminary studies also showed loss of structure in guadinine HCl. The circular dichroism spectrum of the polymer exhibited a deep trough at 190 nm. The marked similarities of solution properties of (Pro-Pro-βAla)_n to (Pro-Pro-Gly)_n suggest that β-alanine can replace glycine in generating collagen-like helix in solution.     

A mathematical model of biological evolution

In order to understand generally how the biological evolution rate depends on relevant parameters such as mutation rate, intensity of selection pressure and its persistence time, the following mathematical model is proposed: dN _n (t)/dt=(m _n (t-)N _n (t)+N _n-1(t) (n=0,1,2,3...), where N _n (t) and m _n (t) are respectively the number and Malthusian parameter of replicons with step number n in a population at time t and is the mutation rate, assumed to be a positive constant. The step number of each replicon is defined as either equal to or larger by one than that of its parent, the latter case occurring when and only when mutation has taken place. The average evolution rate defined by is rigorously obtained for the case (i) m _n (t)=m _n is independent of t (constant fitness model), where m _n is essentially periodic with respect to n, and for the case (ii) (periodic fitness model), together with the long time average m of the average Malthusian parameter . The biological meaning of the results is discussed, comparing them with the features of actual molecular evolution and with some results of computer simulation of the model for finite populations.An early version of this study was read at the International Symposium on Mathematical Topics in Biological held in kyoto, Japan, on September 11–12, 1978, and was published in its Procedings.     

Block oligopeptides (L-lysyl)m-(L-alanyl) L-tyrosyl-(L-alanyl)n- (L-lysyl)m. I. Synthesis through fragment condensation and characterization

Model peptides containing one aromatic residue were synthesized and characterized in order to investigate their interactions with polynucleotides. Chromatographically pure block oligopeptides (L -alysyl)_m-(L -alanyl)_n- L -tyrosyl- (L -alanyl)_n, with n = 3 and m=3 or 6, were prepared by fragments condensation using the mixed anhydride method. The protected fragments were prepared by stepwise addition of amino acid residues through the dicyclohexylcarbodiimide method. The purity of the intermediate coupling product was analyzed by gradient elution chromotography on carboxylmethylcellulose. Both block oligopeptides were isolated by preparative chromatography on carboxymethylcellulose. The different features of these syntheses are discussed.     

Synthesis of Several Fructo-oligosaccharides by Asparagus Fructosyltransferases

Nine fructo-oligosaccharides, synthesized in vitro from sucrose by an enzyme preparation from asparagus roots, were isolated and their structures were elucidated to be 1^F (1-β-fructofuranosyl)_n sucrose [n = 1 (1-kestose), 2 (nystose) and 3], 6^G (1-β-fructofuranosyl)_n sucrose [n=1 (neokestose), 2 and 3] and 1^F (1-β-fructofuranosyl)_m-6^G (1-β-fructofuranosyl)_n sucrose [m=1, n=1; m=2, n =1; and m =1, n=2]. These saccharides are all known to occur naturally in asparagus roots, but 6^G (1-β-fructofuranosyl)₃ sucrose and 1^F (1-β-fructofuranosyl)_m-6^G-(1-β-fructofuranosyl)_n sucrose (m=1, n =1; and m=1, n=2) were the first saccharides enzymatically synthesized in vitro. Also three types of fructosyltransferases were presumed to be involved in the biosynthesis of these oligosaccharides in asparagus roots.     

Polymerization of DL-phenylalanine NCA initiated by copolymer of sarcosine and DL-phenylalanine

Polymerizations of DL -phenylalanine NCA by block copolymers of sarcosine and DL -phenylalanine, designated by (Phe)_m(Sar)n and capable of reaction at the phenylalanyl terminal, were investigated in nitrobenzene solution at 25°C. With increasing n for constant m (m = 0, 1, 2, and 5), the polymerization rate greatly increased. Previously the acceleration of the initiation reaction in the polymerization of DL -phenylalanine NCA by polysarcosine (m = 0) was reported. The present results showing the acceleration by the copolymers of sarcosine and DL -phenylalanine indicate the presence of the polymer effect in the propagation reaction as well. However, the polymer effect was most marked with polysarcosine (m = 0), and decreased with increasing m. The same polymerizations by sequential copolymers composed of ten sarcosine units and two DL -phenylalanine units were also investigated. Again with these copolymer catalysts the polymerization rate was larger than that by monomeric amines. But the polymer effect decreased sharply when the phenylalanine units take positions near the terminal amine group of the copolymer catalyst. These two deteriorating effects of the phenylalanine unit have been interpreted in terms of the decrease of the flexibility of polymer chain, caused possibly by an intramolecular hydrogen bond of the phenylalanine unit.     

Thermodynamics of base interaction in (A)n and (A.U)n

Using precision scanning microcalorimetry we studied (A)_n and (A·U)_n melting in highly diluted solutions (0.3 to 5.0 mm) with different Na⁺ activity. This permitted us to determine directly the thermodynamic functions of stacking interaction in (A)_n and base-pairing in (A·U)_n. For (A-A) stacking at (A)_n melting temperature we obtained ΔH^(A)n_m = 12.6 kJ mol^?1; ΔS^(A)n_m = 41 J K^?1 mol^?1. For A·U base-pairing at a standard temperature of 298 K and 0.1 m-Na⁺ we have: ΔH^(A·U) = 34 kJ mol^?1; ΔS^(A·U) = 102 J K^?1 mol^?1ΔG^(A·U) = ?3.5 kJ mol^?1.     

Studies of DNA dumbbells. IV. Preparation and melting of a DNA dumbbell with the 16 base-pair sequence 5′G-T-A-T-C-C-C-T-C-T-G-G-A-T-A-C3′ linked on the ends by dodecyl chains

The preparation and melting of a 16 base-pair duplex DNA linked on both ends by C¹²H²⁴ (dodecyl) chains is described. Absorbance vs temperature curves (optical melting curves) were measured for the dodecyl-linked molecule and the same duplex molecule linked on the ends instead by T₄ loops. Optical melting curves of both molecules were measured in 25, 55, and 85 mM Na⁺ and revealed, regardless of [Na ⁺], the duplex linked by dodecyl loops is more stable by at least 6°C than the same duplex linked by T₄ loops. Experimental curves in each salt environment were analyzed in terms of the two-state and multistate theoretical models. In the two-state, or van't Hoff analysis, the melting transition is assumed to occur in an all-or-none manner. Thus, the only possible states accessible to the molecule throughout the melting transition are the completely intact duplex and the completely melted duplex or minicircle. In the multistate analysis no assumptions regarding the melting transition are required and the statistical occurrence of every possible partially melted state of the duplex is explicitly considered. Results of the analysis revealed the melting transitions of both the dodecyl-linked molecule and the dumbbell with T₄ end loops are essentially two state in 25 and 55 mM Na⁺. In contrast, significant deviations from two-state behavior were observed in 85 m MNa⁺. From our previously published melting data of DNA dumbbells with T_n end loops where n = 2, 3, 4, 6, 8, 10, 14 [T. M. Paner, M. Amaratunga, and A. S. Benight, (1992) Biopolymers, Vol. 32, pp. 881–892] and the dumbbell with T₄ end loops of this study, a plot of d(T_m)/d ln [Na⁺] was constructed. Extrapolation of this data to n = 1 intersects with the value of d (T_m)/d ln [Na⁺] obtained for the alkyl-linked dumbbell, suggesting the salt-dependent stability of the alkyl-linked molecule behaves as though the duplex of this molecule were linked by end loops comprised of a single T residue. © 1993 John Wiley & Sons, Inc.     

Pressure- and parathyroid-hormone-dependent Ca2+ transport in rabbit connecting tubule: Role of the stretch-activated nonselective cation channel

To characterize the Ca²⁺ transport process across the apical membrane of the rabbit connecting tubule (CNT), we examined the effects of luminal pressure on parathyroid hormone (PTH)-dependent apical Ca²⁺ transport in this segment perfused in vitro. An increase of perfusion pressure (0.2 to 1.2 KPa) caused cytoplasmic free Ca²⁺ concentration ([Ca²⁺].) to increase by 42 ± 11 nm in Fura-2 loaded perfused CNT. The response was accentuated when 10 nm PTH was added to the bath (101 ± 30 nm, n = 6). Addition of 0.1 mm chlorphenylthio-cAMP (CPT-cAMP) to the bath also augmented the [Ca²⁺]; response to pressure from 36 ± 16 to 84 ± 26 nm (n = 3). Under steady perfusion pressure at 1.2 KPa, PTH (10 nm) increased [Ca²⁺]; by 31 ± 7 nm (n = 5), whereas it did only slightly by 6 ± 2 nm (n = 12) at 0.2 KPa. The pressure-dependent increase of [Ca²⁺]; was abolished by removing luminal Ca²⁺ (n = 3), and was not affected by 0.1 and 10 m nicardipine (n = 4) in the presence of 10 nm PTH. Cell-attached patch clamp studies on the apical membrane of everted CNT with pipettes filled with either 200 mm CaCl₂ or 140 mm NaCl revealed channel activities with conductances of 42 ± 2 pS (n = 4) or 173 ± 7 pS (n = 5), respectively. An application of negative pressure (–4.9 KPa) to the patch pipette augmented its mean number of open channels (NP ₀ ) from 0.005 ± 0.001 to 0.022 ± 0.005 in the Ca²⁺-filled pipette, and was further accelerated to 0.085 ± 0.014 (n = 3) by 0.1 mm CPT-cAMP. In the Na⁺-filled pipette, similar results were obtained (n = 3), and CPT-cAMP did not activate the stretch-activated channel in the absence of negative pressure (n = 3). These results suggest that a stretch-activated nonselective cation channel exists in the apical membrane of the CNT and that it is activated by PTH in the presence of hydrostatic pressure, allowing entry of Ca²⁺ transport from the apical membrane.We appreciate Ms. Hisayo Hosaka and Ms. Yuki Oyama for their technical assistance and Ms. Keiko Sakai for her secretarial work. This research was supported by grants from the Ministry of Education and Culture of Japan (No. 05670054) and from Yamanouchi Foundation for Research on Metabolic Disorders (1992–1993).     

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.     

Chemically reacting systems of the type A + B ⇄ AB. II. Osmometry

The osmotic pressure equation for nonideal, associating systems of the type nA +mB ? A_nB_m, has been derived, by using the assumption yA _nB _m/yA ⁿyB ^m = 1. This treatment can also be applied to related associations such as nA + mB ? AB + AB₂ + A₂B + …. From osmotic pressure experiments on the pure reactants it is possible to obtain the molecular weights (M_A and M_B) of the reactants and also the virial coefficients (B_AA and B_BB) of the reactants. The osmotic pressure of a nonreacting mixture of A and B can be calculated from these measurements. It can be used along with osmotic pressure measurements on equilibrium mixtures of A and B to obtain expressions containing the equilibrium constant (or constants) and the cross-virial coefficients (B_AB and B_BA). Several procedures are described for the evaluation of the equilibrium constant (or constants) and the B_AB or B_BA terms. It appears that this procedure is a general one which is applicable to associations of the type nA + mB ? AB + A₂B + AB₂ + …. By correcting for nonideal behavior, one should then be able to apply it to any method available for analyzing ideal associations of the types considered here. In addition it is possible, subject to certain restrictions, to analyze associations of the type 3A + B ? A₂ + AB.