Conformational studies on the pyridoxal Schiff bases of polypeptides

The pyridoxal Schiff bases of the polypeptides poly(L -lysine), poly(L -ornithine), and poly(L -α,γ-diaminobutyric acid) were prepared and investigated in water/methanol by CD spectra and equilibrium dialysis experiments. Only the poly(L -α,γ-diaminobutyric acid) derivative is characterized by a relevant optical activity similar to that found in pyridoxal enzymes. The stereospecific interactions between the pyridoxylideneimine group and the polypeptide chain prevent the hydrolysis reaction of the aldimine bond.     

Metal complexs of poly (α-amino acids): Cu(II) chelates of acetoacetylated poly(L-lysine), poly(L-ornithine), and poly(L-diaminobutyric acid)

The cupric complexes of poly(N^ε-acetoacetyl-L -lysine), [Lys(Acac)]_n′ poly(N^δ-acetoacetyl-L -ornithine), [Orn(Acac)]_n′ and poly(N^γ-acetoacetyl-L -diaminobutyric acid), [A₂bu-(Acac)]_n, as well as of the model compound n-hexyl acetoacetamide, have been investigated by means of absorption, potentiometric, equilibrium dialysis, and CD measurements. While in the complex of the model compound, one chelating group is bound to one cupric ion, in the polymeric complexes two β-ketoamide groups are bound to Cu(II) under the same experimental conditions. The binding constant of cupric ions to the three polymers and the formation constant of the Cu(II)-nhexylacetoacetamide complex have been evluated. Investigation on the chiroptical properties of the three polymeric complexes shows that the peptide backbone does not undergo conformational transitions, remaining α-helical when up to 20% of the side chains are bound to Cu(II). The optical activity of the β-ketoamide chromophores is substantially affected by complex formation and is discussed in terms of asymmetric induction from the chiral backbone.     

Ferric complexes of acetoacetyl derivatives of poly(L-lysine), poly(L-ornithine), and poly(L-diaminobutyric acid). II. Conformational properties in solution. Evidence for a stereospecific complex formation

The conformational properties of ferric complexes of poly(N^ε-acetoacetyl-L -lysine), poly(N^δ-acetoacetyl-L -ornithine), and poly(N^γ-acetoacetyl-L -diaminobutyric acid) were investigated in 1:1 water/dioxane by CD techniques. Optical activity was found in the visible and in the uv absorption region of the polymeric complexes. The conformation of the peptide backbone was always that of a right-handed α-helix, and was found independent of the degree of complexation, at least up to a degree of binding of 20%. In the absorption region of the side-chain chromophores the optical activity is substantially affected by complex formation. In all three cases a splitting of the ligand π → π* transition centered at 257 nm is observed. These data suggest a stereospecific complex formation. From the signs of the splitting it also appears that the chirality of the poly(N^δ-acetoacetyl-L -ornithine) complex is opposite that of the other two polymers.     

Interactions between mucopolysaccharides and cationic polypeptides in aqueous solution: Chondroitin 4-sulfate and dermatan sulfate

Circular dichroism spectroscopy has been used to study the interactions of both dermatan sulfate and chondroitin 4-sulfate with the cationic polypeptides; poly(L -arginine), poly(L -lysine), and poly(L -ornithine). The results indicate that the mucopolysaccharides have a conformation directing effect on both poly(L -arginine) and poly-(L -lysine) such that these polypeptides adopt the α-helical conformation. The extent of interaction in each polypeptide-polysaccharide system can be judged by the degree of induced helicity and the “melting temperature” at which the interaction is disrupted On comparison of these results with those previously obtained for chondroitin 6-sulfate-polypeptide mixtures, the extent of interaction can be seen to depend on the length of the amino acid side chain and the positions of the anionic groups on the mucopolysaccharide chain. Such considerations place the three mucopolysaccharides in order of increasing interaction: chondroitin 4-sulfate < chondroitin 6-sulfate < dermatan sulfate. These results are correlated with observations that dermatan sulfate is bound more tightly to collagen in connective tissues than are the other two polysaccharides.     

Conformation of poly(L-arginine). II. Complexes with polyanions

Conformaitons of poly(L -arginine)/polyanion complexes were studies by CD measurements. The polyanions were the homoplolypeptides poly(L -glutamic acid) and poly(L -aspartic acid); the synthetic polyelectrolytes and polyethylenesulfonate; and the polynucleotides were native DNA, denatured DNA, and poly(U). It was found that poly(L -arginine) forms the α-helical conformation by interacting with the acidic homopolypeptides and the synthetic anionic polyelectrolytes. In each complex, poly(L -glutamic acid) is in the α-helical conformation, whereas poly(L -aspartic acid) is mostly in the random structure. The poly(L -glutamic acid) complex changed into the β-sheet structure at the transition temperature about 65°C in 0.01M cacodylate buffer (pH 7). Even in the presence of 5M urea, this complex remained in the α-helical conformation at room temperature. The existence of the stable complex of α-helical poly(L -arginine) and α-helical poly(L -glutamic acid) was successfully supported by the model building study of the complex. The α-helix of poly(L -arginine) induced by binding with polyacrylate was the most stable of the poly(L -arginine)-polyanion complexes examined as evidenced by thermal and urea effects. The lower helical content of the polyethylenesulfonate-complexed poly(L -aginine) was explained in terms of the higher charge density of the polyanion. On the other hand, native DNA, denatured DNA, and poly(U) were not effective in stabilizing the helical structure of poly(L -arginine). This may be due to the rigidity of polyanions and to the steric hindrance of bases. Furthermore, the distinitive structual behavior of poly(L -arginine) and poly(L -lysine) regarding polyanion interaction has been noticed throughout the study.     

Stability of helical polyamino acids in solution at high temperatures

Optical rotatory dispersion studies have been carried out at temperatures up to 150 °C. on poly(γ-benzyl L -glutamate) in α-chloronaphthalene and N-methylcaprolactam, and on poly-ε-carbobenzoxy-L -lysine, poly-δ-carbobenzoxy-L -ornithine, and poly(L -glutamic acid) in N-methylacetamide. The Moffitt-Yang ?b₀ values were large in all cases, but significant decreases in ?b₀ were observed at the upper temperature limits of the study suggesting that a transition region was being entered. Polymer degradation generally precluded examination of the systems through the suggested transition region.     

Induced CD and interaction of some porphyrin derivatives with α-helical polypeptides in aqueous solutions

Absorption spectra and induced CD have been measured on aqueous solutions of water-soluble porphyrins with α-helical poly(L -glutamic acid) or α-helical poly (L -lysine) at different mixing ratios. For the former, porphyrin is porphine-meso-tetra (4-N-methylpyridinium) (TMpyP), and for the latter, it is porphine-meso-tetra (4-benzenesulfonate) (TPPS) or porphine-meso-tetra(4-benzoate) (TPPC). All the solutions of porphyrin-polypeptide systems show hypochromism in the Soret band and induced CD in the Soret region. The CD is characterized by a positive band at a shorter wavelength and a stronger negative band at a longer wavelength. The hypochromicity and the magnitude of molar ellipticities are much larger for the TPPS– and TPPC–poly (L -lysine) systems than for the TMpyP–poly (L -glutamic acid) system. Porphyrin ions bind to the α-helix electrostatically, and the two components of the Soret transition of porphyrin are subject to dissymmetric perturbation. TMpyP ions bind to the α-helix at isolated sites, while TPPS ions and TPPC ions are in pairs on the α-helix, that is, two ions bind consecutively and dissymmetrically. In the TMpyP–poly (L -glutamic acid) system a single CD band is associated with each of the two components of the Soret transition, and these are of opposite sign. In the TPPS– and TPPC–poly (L -lysine) systems, a pair of positive and negative CD bands is associated with each of the two components, thus giving apparently a single pair of CD bands with a shoulder, owing to partial overlapping.     

Comparison of helix stabilities of poly-L-lysine, poly-L-ornithine, and poly (L-diaminobutyric acid)

The helix–coil transitions of aqueous solutions of poly-α-L -lysine (PLL), poly-α-L -ornithine (PLO), and poly(α,γ-L -diaminobutyric acid) (PLDBA) have been investigated as functions of pH at 25°C and of temperature at pH 11.75, where these polymers are uncharged; in the cases of the latter two polyamino acids, the transitions have also been studied as functions of apparent pH in methanol-water solution (50/50 by volume). The helix stability of the polypeptides is shown to be a direct function of the number of methylene groups on the side chain. From an analysis of potentiometric titration data, we find that the difference between the helix stability of PLL and that of PLO is due to a difference of about 1 e.u. in the ΔS° of the transition. Combining the “melting curves” obtained from optical rotatory dispersion studies with the potentiometric titration data permits evaluation of the initiation parameter Z (or 1/σ^½) of the statistical mechanical theories for these transitions. The value obtained for Z in the case of uncharged aqueous PLO is ca. 35.     

Anisotropic rotational motions of poly(L-glutamic acid) in the alpha-helix conformation

The anisotropic rotational motion of the backbone and the side chains of poly(L -glutamic acid) in the α-helical structure was investigated using the ¹³C-T₁ and T₂ relaxation times of all carbon atoms with directly attached protons, obtained at a ¹³C-Larmor frequency of 67.89 MHz. The evaluation of the nmr data was carried out according to the previously derived anisotropic diffusion model, in which the macromolecule is considered a rigid rod. The rotation of the backbone is characterized by two diffusion constants, D₁ and D₃, describing the rotation perpendicular to and around the symmetry axis. The additional internal motion of the C^β-methylene group is described as a jump process with a jump rate, k₁, between two allowed rotametric states. Steric considerations indicate that the occupation of the third rotameric position is forbidden. The rotation of the C^γ-methylene group is decribed as a one-dimensional diffusion process around the C^β–C^γ bond. Investigation of the temperature dependence of the relaxation parameters led to the temperature dependence of the dynamic parameters. Activation energies were determined from these data. The dynamic parameters obtained for poly(L -glutamic acid) at 291 K are compared with the corresponding results of a previous study of poly(L -lysine). The development of an anisotropic diffusion model for the motions of the rod-shaped poly(L -lysine) α-helix and its application to the interpretation of the ¹³C-relaxation data of this molecule have already been published previously. In this model, both the overall molecular tumbling and the various internal motions have been characterized by diffusion constants or jump rates typical for each process. These dynamic parameters can be calculated from the spin–lattice relaxation times, the spin–spin relaxation times and the NOE factors of the C^α, C^β, and C^γ nuclei of the polypetide. In the present paper, we describe the application of the above-mentioned dynamic model to the interpretation of ¹³C-relaxation studies of a further homopolypeptide, poly(L -glutamic acid), in the α-helical structure. Furthermore, we studied the temperature dependence of the relaxation times of this polymer and determined the anisotropic diffusion parameters at each temperature. From their temperature dependence and from comparison of our present results with the data of our previous study of poly(L -lysine), we were able to derive new insights into the intramolecular diffusion processes and the excitation of various motions.     

Conformational studies of sequential polypeptides containing lysine and tyrosine

The conformation of three sequential copolypeptides, poly(L -tyrosyl-L -lysine), poly(L -tyrosyl-L -lysyl-L -lysine), and poly[L -tyrosyl-(L -lysyl)₂-L -lysine] have been studied by a variety of techniques, including CD, ir spectroscopy, analytical ultracentrifugation, and x-ray diffraction. Depending upon the pH and sovent composition, poly(L -tyrosyl-L lysyl-L -lysine) and poly [L -tyrosyl-(L lysyl)₂-L -lysine] can adopt either the α-helical or random-coil conformation, while poly(L -tyrosyl-L -lysine) forms either inter- or intramolecular β-structures.     

Ferric complexes of acetoacetyl derivatives of poly(L-lysine), poly(L-ornithine), and poly(L-diaminobutyric acid). I. Preparation and absorption properties in solution

Poly(N^ε-acetoacetyl-L -lysine), poly(N^δ-acetoacetyl-L -ornithine) and poly(N^γ-acetoacetyl-L -diaminobutyric acid) form colored complexes with ferric ions in water/dioxane solutions. These complexes are soluble at pH values lower than 2.8 and show their maximum absorption at 257 nm in the uv and at 478 nm in the visible region; whereas the ferric complex of the model compound n-hexylacetoacetamide exhibits absorptions centered at 258 and 536 nm, respectively. It is shown that in the complex of the model compound one metal ion is bound per acetoacetamide group, while in the complexes of the three polymers two β-ketoamides side chains are bound per ferric ion under the same solvent, pH, concentration, and ionic strength conditions. The binding constants of ferric ions to the three polymers, and the formation constant of the ferric complex of the model compound are also evaluated.     

The thermal induced helix--beta transition of poly(N epsilon-methyl-L-lysine) and poly(N delta-ethyl-L-ornithine) in aqueous solution

Uncharged poly(N^ε-methyl-L -lysine) (PMLL) and its isomer, poly(N^δ-ethyl-L -ornithine) (PELO), in alkaline solution (pH ca. 12) undergo a helix-to-β transition upon mild heating at 50°C or higher in a manner similar to that of poly(L -lysine) (PLL). The rate of conversion follows the order: PMLL < PELO < PLL. The helix can be regenerated upon cooling near zero degrees, for instance, after more than 12 hr at 2°C. At concentrations less than 0.02% the β form is intramolecular, but at higher concentrations both intra- and intermolecular β forms are generated. Poly(N^δ-methyl-L -ornithine) (PMLO), an isomer of PLL, behaves like poly(L -ornithine); uncharged PMLO in alkaline solution is partially helical and becomes disordered at elevated temperatures.     

Intensities and other spectral parameters of infrared amide bands of polypeptides in the alpha-helical form

Intensities and other spectral parameters of infrared amide I and II bands of α-helical polypeptides in solutions have been determined for poly(γ-benzylglutamate), poly(γ-ethylglutamate), and polymethionine in chloroform, polylysine, poly(glutamic acid), and fibrillar protein tropomyosin from rabbit muscles in heavy water. The majority of spectral parameters are characteristic. The half-width of the amide I band was found to vary in the range of 15–40 cm^?1 for different polypeptides in the different solutions. The correlation between this parameter of the amide I band and the stability of the α-helix was estimated. A new weak band near 1537 cm^?1 of unknown origin was observed for the hydrogen form of polypeptides in the α-helical state.     

Conformations of poly-L-valine in solution

In order to test theoretical predictions that poly-L -valine can exist in an α-helical conformation, water-soluble block copolymers of L -valine and D , L -lysine were prepared. By carrying out the synthesis on a resin support (with the use of N-carboxyanhydrides) contamination of the individual blocks by any unreacted monomer from the previous block was avoided. A single glycine residue was incorporated at the C-terminus of the chain for use in amino acid analyses. Using optical rotatory dispersion and circular dichroism criteria, about 50% of the short valine block of (D , L -lysine HCl)₁₈-(L -valine)₁₅-(D , L -lysine-HCl)1₆-glycine was found to be in the right-handed α-helical conformation in 98% aqueous methanol, in water, the polymer appears to be a dimer, with the valine block being involved in the formation of an intermolecular β-structure.     

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

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

Optical Rotatory Dispersion of Polypeptides in the Near-Infrared Region

For the first time ORD measurements in the near-infrared region from 0.7 to 2.0 μ for well-known polypeptides, namely, poly(γ-benzyl L -glutamate), poly(L -glutamic acid), poly-L -lysine·HCl, poly-S-carbobenzoxymethyl-L -cysteine, and Bombyx mori silk fibroin, were carried out. It was found that the value of the optical activity infrared term, which is proportional to the sum of rotational strengths of vibrational transitions, depends on polypeptide conformation. The optical activity infrared term value is equal to zero for the random-coil conformation, it is small but exceeds the measurement error for the α-helical state, and finally, for the β conformation it is an order of magnitude higher than for the α-helical state. The obtained results permit one to hope that on the basis of ORD measurement in the near-infrared region it will be possible to suggest a method of determining the β-form content in polypeptides and proteins     

Studies on the Bacterial Formation of a Peptide Antibiotic,Colistin

The biosynthetic pathway of α,γ-diaminobutyric acid, 6 moles of which are involved in the colistin molecule as a main component, was investigated. On the basis of the isotopic results using aspartic acid-U-¹⁴C as a precursor and also the finding of transaminase activity between α-ketog?utaric acid and α,γ-diaminobutyric acid, though in reverse reaction, α,γ-diaminobutyric acid was proved to be synthesized from aspartic acid via aspartyl-phosphate and aspartic β-semialdehyde. α,γ-Diaminobutyric acid did not inhibit asparto-kinase activity of this bacterium, the first enzyme involved in the process of α,γ-diamino-butyric acid synthesis from aspartic acid, while the end product amino acids such as lysine, threonine and methionine showed inhibition for aspartokinase activity.

On the other hand, α,γ-diaminobutyric acid might be rate-limiting factor in colistin formation, because of stimulatory effect of this diamino acid when added to the medium on colistin production. Furthermore, colistin production appeared to be related with the defect of TCA-cycle and further the resultant increase in activities of the key enzymes such as isopropylmalate synthetase, α-acetolactate synthetase and aspartokinase involved in the biosynthetic pathways of valine, leucine and isoleucine, respectively.     

Interaction of sodium decyl sulfate with poly(L-ornithine) and poly(L-lysine) in aqueous solution

The binding isotherms of sodium decyl sulfate to poly(L -ornithine), poly(D ,L -ornithine), and poly(L -lysine) at neutral pH were determined potentiometrically. The nature of a highly cooperative binding in all three cases suggests a micelle-like clustering of the surfactant ions onto the polypeptide side groups. The hydrophobic interaction between the nonpolar groups overshadows the coulombic interaction between the charged groups. The titration curves can be interpreted well by the Zimm–Bragg theory. The average cluster size of bound surfactant ions is sufficiently large to promote the β-structure of (L -Lys)_n even at a very low binding ratio of surfactant to polypeptide residue, whereas the onset of the helical structure for (L -Orn)_n begins after about 7 surfactant ions are bound to two turns of the helix. The CD results are consistent with this explanation.     

Electric field effect on the helix–coil transition of poly(L-α,γ-diaminobutyric acid hydrochloride) in methanol/water mixtures

Transient electric birefringence of poly(L -α,γ-diaminobutyric acid hydrochloride) in methanol/water mixtures has been measured over a wide range of field strengths and solvent compositions and at different polymer concentrations and temperatures. The molar ellipticity at 222 nm and the specific Kerr constant underwent an abrupt change between 75 and 80 vol % methanol at 25°C, accompanied by a solvent-induced helix–coil transition. Anomalous birefringence transients were observed between 78 and 80 vol% methanol above threshold field strengths. The double logarithmic plots of the steady-state specific birefringence versus the square of field strength for different solvent compositions and polymer concentrations could be superimposed by shifting them horizontally along the abscissa and vertically along the ordinate except for the range where anomalous transients were observed. The threshold field strength could be estimated from the point at which a downward deviation occurred. It increased with increasing polymer concentration and with increasing methanol content on the verge of the transition region. The results were interpreted as indicating that a conformational change from the charged helix to the charged coil is induced by high fields in this system, as in the case of poly(L -lysine hydrobromide) in methanol/water mixtures.     

Syntheses of poly[γ-(l)-menthyl L- and D-glutamates] and their secondary structures

γ-(l)-Menthyl L - and D -glutamates were prepared by a fusion reaction of N-phthalyl-L - and D -glutamic anhydrides with l-menthol, followed by hydrazinolysis. The monomers were polymerized to poly[γ-(l)-menthyl L - and D -glutamates] by the N-carboxyanhydride method. These polymers were soluble in many organic solvents, such as ethyl ether, chloroform, tetrahydrofuran, and n-hexane. From the results obtained by a study of the infrared absorption spectra, the x-ray photographs, the optical rotatory dispersions and the circular dichroisms, poly[γ-(l)-menthyl L -glutamate] was found to be a right-handed α-helix in the solid state and in solution. Similarly, poly[γ-(l)-menthyl D -glutamate] was a left-handed α-helix. The helix-coil transition of these polymers was observed in the vicinity of 40% dichloroacetic acid in a chloroform–dichloroacetic acid mixture.