Solution properties of synthetic polypeptides. VI. Helix-coil transition of poly-N5-(3-hydroxypropyl)-L-glutamine

A new procedure for evaluating u and σ characterizing σ-helix-forming polypeptides in solution was derived from Nagai's theory for the helix–coil transition of such polymers. Here u is the activity for helix formation from random coil, and σ is the helix initiation parameter. The necessary data are the helical content f_N at fixed solvent and temperature as a function of N, where N is the degree of polymerization of the polypeptide sample. Such data were obtained from ORD measurements on a number of fractionated samples of poly-N⁵-(3-hydroxypropyl)-L -glutamine (PHPG) in mixtures of water and methanol covering the complete range of composition and at various termperatures (5–40°C). When analyzed in terms of the proposed procedure, they yielded values of σ which were in the range (3.2 ± 0.6) × 10^?4, substantially independent of solvent composition and temperature. These values were much larger than those obtained recently for σ of poly(β-benzyl-L -aspartate) in m-cresol and in a mixture of chloroform and DCA. The data for [η] and s₀ (limiting sedimentation coefficient) as functions of molecular weight indicated that the molecular shape of PHPG in pure methanol is essentially rodlike, whereas that in pure water is not entirely randomly coiled but rather may be regarded as an interrupted helix. These indications were consistent with the results from ORD measurements. When plotted against the corresponding values of f_N, the values of [η] and [s₀] for PHPG in mixtures of water and methanol of various compositions and temperatures formed smooth composite curves, and we attributed these phenomena to the fact that σ of PHPG was nearly constant under these solvent conditions. Here [s₀] stands for a reduced limiting sedimentation coefficient which is equal to the inverse friction factor of the solute molecule.     

Solution properties of synthetic polypeptides. 18. Helix-coil transition of poly-N5-(2-hydroxyethyl)L-glutamine

The helix–coil transition of poly-N⁵-(2-hydroxyethyl)L -glutamine (PHEG) in aqueous isopropanol was examined by means of optical rotatory dispersion (ORD) and intrinsic viscosity [η] measurements. The Zimm–Bragg parameters σ and s for the transition were determined from the ORD data as a function of molecular weight. It was found that the transition was characterized by a relatively low cooperativity; the values of \documentclass{article}\pagestyle{empty}\begin{document}$ \sqrt \sigma $\end{document} were in the range from 0.039 to 0.066, depending on the solvent composition. These σ values are much larger than those reported for other polypeptide–solvent systems. The transition enthalpy was negative and its magnitude varied with the solvent composition, with a maximum of 620 cal/mol at 40 wt% isopropanol. The curve of [η] versus helical content for a high-molecular-weight sample exhibited a very broad minimum, and this behavior was attributed to the low cooperativity of the transition.     

Solution properties of synthetic polypeptides. V. Helix–coil transition in poly(β-benzyl L-aspartate)

Simple approximate expressions have been derived from the theory of Zimm and Bragg for use in the analysis of experimental data on the helix-coil transition in polypeptide. On the basis of the resulting expressions practical procedures are proposed to determine two basic parameters characterizing a thermally induced transition, i.e., helix initiation parameter σ and enthalpy change for helix formation, ΔH. They have been applied to the data for poly(β-benzyl L -aspartate) (PBLA) with the result: σ = 1.6 × 10^?4 and ΔH = ?450 cal/mole for PBLA in m-cresol; σ = 0.6 × 10^?4 and ΔH = 260 cal/mole for PBLA in chloroform containing 5.7 vol-% of dichloroacetic acid. This result gives evidence that σ may change not only from one polypeptide to another but also for a given polypeptide in different solvents. The change in limiting viscosity number [η] accompanying the transition was measured in the same solvents. The curve of [η] versus helical content had a relatively monotonic shape for the chloroformdichloroacetic acid solutions as compared with that for the m-cresol solutions, indicating that [η] depended largely on σ. Provided that [η] is a direct measure of the mean-square radius of gyration, 〈S²〉, the results are consistent with the theoretical predictions of Nagai and of Miller and Flory for 〈S²〉.     

Helix-coil transition of poly(gamma-benzyl-l-glutamate) in the dioxane-dichloracetic acid mixture.

The thermal helix–coil transition of four samples of poly(γ-benzyl-L -glutamate) in the dioxane–dichloroacetic acid (DCA) mixture was studied by optical rotatory dispersion method. The transition occurred at very high DCA content (97.5% by weight) in this system. The transition parameters have been evaluated by two methods based on the theories of Zimm and Bragg and of Nagai. The values of the helix initiation parameter and of the enthalpy of transition were found to be (4.4 ± 0.4) × 10^?5 and 340 cal/mole, respectively. The range of validity and equivalence of the two methods is discussed.     

Solution properties of synthetic polypeptides. XII. Enthalpy changes accompanying helix-coil transition of polypeptide

For helix-coil transitions of polypeptide in binary mixtures consisting of helix-forming solvent and coil solvent, the transition enthalpy ΔH(T,x) has been found to depend significantly on temperature (T) and solvent composition (x). For such systems, calorimetric measurements may yield some averages of ΔH(T,x) which are no longer amenable to direct comparison with ΔH itself. Theoretical equations relating calorimetric data to ΔH(T,x) are derived and tested favorably with experimental data. It is demonstrated that the transition enthaply from heat capacity measurements is approximately equal to ΔH_cfm, while those from heat of dilution and heat of solution measurements are equal to ΔH_c. Here ΔH_c denotes the value of ΔH at the transition point and f_m represents the maximum helical content attained in a thermally induced transition. The discrepancies among calorimetric data are also discussed.     

Solution properties of synthetic polypeptides. XVII. Stability of the alpha-helical conformation of poly (gamma-benzyl L-glutamate) in helicogenic solvents

The Zimm–Bragg parameters s and σ were determined for poly(γ-benzyl L -glutamate) (PBLG) in m-cresol and in dimethylformamide (DMF) from ORD data as a function of molecular weight. It was found that, within the temperature range between 10 and 55°C and on the average, s = 1.6₁ ± 0.1 and √σ = 0.04 ± 0.01 in m-cresol and s = 1.6₅ ± 0.05 and √σ = 0.045 ± 0.015 in DMF. The values of s in m-cresol decreased with increasing temperature, while the values of σ in the same solvent increased. This result for s suggests that PBLG in m-cresol will undergo a thermal helix–coil transition of normal type. The parameters in DMF showed no appreciable trend to vary with temperature. Aside from the difference between the two solvents, our results are consistent with existing data for various conformation-dependent properties such as light-scattering radius, intrinsic viscosity, and dipole moment, each indicating that the polypeptide chain has some flexibility in helicogenic solvents.     

Biodegradability of synthetic branched polypeptide with poly(L-lysine) backbone

A detailed investigation is reported about the biodegradation of poly[Lys(DL-Alam)], m approximately 3, (AK) the common inside area of a branched polypeptide model system developed by our group over the last decade. Enzymatic hydrolysis was carried out by the exopeptidase aminopeptidase M, or the endopeptidase trypsin, or their mixture. Ion-exchange column chromatography, paper electrophoresis and thin-layer chromatography were utilised to achieve separation of metabolites. Breakdown products were identified by the aid of synthetic oligopeptides representing the potential fragments (DL-Ala2, DL-Ala3, Lys(DL-Alam), m = 1-3). The kinetics and the degree of enzymatic degradation were determined. The ratio of peptide/amino acid amounts in the hydrolysate was found to be 1.07 after 24 h treatment with aminopeptidase M, 3.0 with trypsin and 1.3 with aminopeptidase - trypsin mixture. The overall results indicated that the proteolysis of AK by an aminopeptidase M and trypsin mixture proceeds stepwise at multiple sites on the polypeptide chain. The degradation is significantly retarded as compared to that of alpha- or epsilon-polylysine. A mechanism of degradation is suggested based on the experimental results.     

Helix-coil equilibria of poly (rA) · poly (rU)

Absorbance melting curves of the double-stranded (rA) · (rU) helix, made with fractionated homopolynucleotides of matched length, have been obtained over a 15-fold range of [Na⁺] and 30° range of temperature. An excellent fit of the observed profiles was obtained with theoretical curves calculated on the basis of the simplest interpretation for the occurrence of particular equilibria [1–3]; the complete molecular partition function being evaluated by the power series method developed by Applequist [4–6]. The stability constant was evaluated from literature values for the calorimetric enthalpy. The loop closure exponent was best represented by 2.22 ± 0.04 for the mismatching loop mode of melting and 1.22 for the matching mode and was independent of [Na⁺] and temperature. Assuming the applicability of the nonintersecting random walk value of 1.9 ± 0.1, these results would suggest a slight bias toward matched loop formation during melting of homopolynucleotides that might be expected to form only mismatched loops. The value of the stacking parameter at 60°C was only ~6% higher than that at 30°C, 0.0221 (0.0184 for the matching case). Calculated melting curves indicate the occurrence of a fifth-order phase transition when the mean helix length is only ~13 base-pairs, or about one full turn of the helix.     

Carrier design: biodistribution of branched polypeptides with a poly(L-lysine) backbone

The biodistribution has been examined in mice of a range of synthetic branched polypeptides which are based on a polylysine backbone but which differ in ionic charge, side-chain structure, and molecular size. Polycationic polypeptides, regardless of their size or primary structure at the branches, were cleared rapidly from the circulation, the liver being the major site of clearance. Polypeptides with glutamic acid in the side chain, which would be amphoteric under physiological conditions, showed a significantly prolonged blood survival, and this was seen with polypeptides in the range of molecular weights of 46,000 up to 213,000. Such polypeptides provide a useful system with which to investigate the effect of structural parameters on the pharmacokinetic properties of carrier molecules and would allow the selection of candidate carriers for a variety of uses.     

Antiviral activity of conjugates between poly(L-lysine) and synthetic oligodeoxyribonucleotides

Short (14 to 20-mer range) synthetic oligodeoxyribonucleotides (oligos) allow to modulate specifically viral or cellular gene expression at various stages thus providing a versatile tool for fundamental studies and a rational approach to antiviral chemotherapy. Several problems, such as metabolic stability and efficient cell internalization of oligos, still limit this approach appreciably, as briefly discussed here. We demonstrate here that the conjugation of 15-mer (beta)-anomeric oligos to poly(L-lysine) allows a specific protection of various cell lines against vesicular stomatitis virus infection at concentrations lower than 1 microM. This can be achieved with oligos complementary to the viral N-protein mRNA initiation site or to viral intergenic sequences, i.e., to untranscribed regions. No antiviral activity can be obtained with (alpha)-anomeric oligos directed against the same targets, although such analogues are much more resistant to nuclease degradation and form stable hybrids, at least in cell-free experiments.     

Helix-coil transition in copolypeptides. I. Poly (gamma-benzyl-co-gamma-methyl L-glutamate)

The helix–coil transition in poly(γ-benzyl L -glutamate-co-γ-methyl L -glutamate) copolypeptides was studied experimentally in nonaqueous solvents and the results compared with theory. It is found that the transition can be described by the same theory as for the homopolypeptides, but the corresponding parameters are not related in the expected way to those of the homopolymers, due to the effect of the side chains on the stability of the helix, which is not taken into account explicitly by the theory.     

15N-NMR spectroscopy. IV. Comparison of poly(L-lysine) and isopoly(L-lysine)

The natural abundance ¹⁵N-nmr spectroscopy has been used to characterize the isomeric polymers (L -Lys)_n and iso (L -Lys)_n in aqueous solution. Although the peptide nitrogens of the two polymers have nearly equivalent shifts at pH < 10, the amino nitrogens differ by 5–6 ppm at pH < 7 and provide an easy means of identification. Furthermore, the polymers are distinguishable by the pK_a of the amino group and the basicity of the peptide nitrogen. At pH 10.3 and 25°C, (Lys)_n exhibits line broadening and an upfield chemical shift of the peptide nitrogen, indicative of the coil → helix transition. The formation of 100% helix may produce a shift as large as 5 ppm, which probably makes ¹⁵N-nmr spectroscopy more suitable for studies of this transition.     

Helix-coil transition in heterogeneous DNA.

The broadening of a helix–coil transition due to base pair heterogeneity is calculated on the basis of a cumulant perturbation expansion in the quasi-grand ensemble. In this ensemble the fictitious, homogeneous chain, to which the perturbation is referred, automatically decreases its correlation length as the heterogeneity increases. This “renormalization” seems to stabilize the perturbation expansion, in view of the good agreement between the present results and the exact theory of a heterogeneous polypeptide helix–coil transition. For the DNA model in which ring entropy is included, the transitions is found to be extremely narrow for an infinite random chain with conventional parameters. A tentative reconciliation of this result with contradictory calculations of some other workers is offered on the basis of end effects, coarse graining, or approximation to the ring entropy. An application of the new method to DNA with a non-random base pair distribution requires evaluation of the correlation function between molecular states (helix or coil), at different sites of the reference chain. The evaluation is reduced to quadrature, but numerical calculations have been made only for the random chain.     

Helix-coil transition of Poly(L-glutamic acid) in N-methylacetamide

The folding of randomly coiled poly(L -glutamic acid) to the helical state has been studied in N-methylacetamide by titration methods. Since this solvent would be expected to form amide-peptide group hydrogen bonds with the unfolded form of the polymer, to a first approximation no helix stabilization could come from intrapolymer hydrogen bonds. The titration data, collected from 30 to 70°C yield the following values per residue for the thermodynamic parameters governing the coil-helix reaction for the uncharged polymer: ΔG_30°C°, ?1. 9 ± 0.1 kcal; Δ H°, 0 ± 0.1 kcal; ΔS_30°C°, 6.3 ± 0.6 eu. In N-methyl acetamide, the helix is an order of magnitude more stable than in water, and this stabilization appears to be entirely the result of the entropy gained by solvent molecules which are released from the polymer upon folding.     

Highly branched poly(L-lysine)

This paper describes the synthesis of several novel water-soluble highly branched polypeptides. The synthesis starts with the ring-opening polymerization of epsilon-benzyloxycarbonyl-l-lysine N-carboxyanhydride (Z-Lys NCA) or epsilon-trifluoroacetyl-l-lysine N-carboxyanhydride (TFA-Lys NCA), followed by end functionalization of the peptide chain with N(alpha),N(epsilon)-di(9-fluorenylmethoxycarbonyl)-l-lysine (N(alpha),N(epsilon)-diFmoc Lys). Deprotection of the N(alpha),N(epsilon)-diFmoc Lys end group affords two new primary amine groups that can initiate the polymerization of a second generation of branches. Repetition of this ring-opening polymerization-end functionalization sequence affords highly branched poly(epsilon-benzyloxycarbonyl-l-lysine) (poly(Z-Lys)) and poly(epsilon-trifluoroacetyl-l-lysine) (poly(TFA-Lys)) in a small number of straightforward synthetic steps. Removal of the side-chain protective groups yields water-soluble and highly branched poly(l-lysine)s, which may be of potential interest for a variety of medical applications.     

Channel structures in synthetic polypeptides with alternating configurations. Conformational analysis of poly(DL-proline)

Theoretical conformational analysis of L,D alternating sequences of poly alpha-amino acids is reported in connection with the ability of naturally occurring peptide and depsipeptide having alternating configurations to increase selectively the ion permeability across membranes. The most stable structures of poly(DL-proline), of which the conformational variability is practically limited to the choice between cis and trans conformations of the peptide bonds, were characterized. The all-trans conformation results in a flat helical structure possessing the main features for acting as an ion channel across membranes as actually found experimentally. Random cis-trans conformational sequences provide an alternative mechanism of ion transport intermediate between the ion channel and the ion carrier.     

Some biophysical and biochemical properties of poly(phthaloyl L-lysine) microcapsules containing hemolysate.

Measurements of oxygen equilibrium, zeta-potential, resistance to flow, carbonic anhydrase activity, and catalase activity were made on sheep erythrocyte hemolysate-loaded poly(phthaloyl L-lysine) microcapsules (artificial red blood cells) prepared by an interfacial polycondensation technique. The measurements revealed that oxygen dissociation equilibrium, zeta-potential, and carbonic anhydrase activity of the microcapsules are almost the same as those of sheep erythrocytes, while the microcapsules have a higher resistance to flow and a lower catalase activity than the erythrocytes. Possible ways of improving the properties of the microcapsules were suggested.     

The alpha-helix to beta-sheet transition in poly(L-lysine): effects of anesthetics and high pressure.

Poly(L-lysine) exists in a random-coil formation at a low pH, alpha-helix at a pH above 10.6, and transforms into beta-sheet when the alpha-helix polylysine is heated. Each conformation is clearly distinguishable in the amide-I band of the infrared spectrum. The thermotropic alpha-to-beta transition was studied by using differential scanning calorimetry. At pH 10.6, the transition temperature was 43.5 degrees C and the transition enthalpy was 170 cal/mol residue. At pH 11.85, the measurements were 36.7 degrees C and 910 cal/mol residue, respectively. Volatile anesthetics (chloroform, halothane, isoflurane and enflurane) partially transformed alpha-helix polylysine into beta-sheet. The transformation was reversed by the application of hydrostatic pressure in the range of 100-350 atm. Apparently, the alpha-to-beta transition was induced by anesthetics through partial dehydration of the peptide side-chains (beta-sheet surface is less hydrated than alpha-helix). High pressure reversed this process by re-hydrating the peptide. Because the membrane spanning domains of channel and receptor proteins are predominantly in the alpha-helix conformation, anesthetics may suppress the activity of excitable cells by transforming them into a less than optimal structure for electrogenic ion transport and neurotransmission. Proteins and lipid membranes maintain their structural integrity by interaction with water. That which attenuates the interaction will destabilize the structure. These data suggest that anesthetics alter macromolecular conformations essentially by a solvent effect, thereby destroying the solvation water shell surrounding macromolecules.