Laser-Raman spectroscopy of biomolecules. XI. Conformational study of poly(L-valine) and copolymers of L-valine and L-alanine

Raman spectroscopic studies have been carried out on polymers of L -valine ranging in degree of polymerization (DP) from 2 to 930. The spectrum of the hexapeptide (DP = 6) is closely similar over the entire range 40–1750 cm^?1 to those of polymers with much higher DP, and the structure is clearly shown to be that of the antiparallel pleated sheet (β-structure) by the amide I and III frequencies. The formation of a little α-helical structure occurs in polymers with DP above 500, although the amount does not appear to be a linear function of DP. The α-helical structure is unstable and readily destroyed in samples cast from trifluoroacetic acid solution. It is stabilized by the incorporation of L -alanine, a strong helix-former; polymers of the latter may in turn be forced into a α-structure in copolymers sufficiently rich in L -valine.     

Block copolymers of amino acids. II. Physicochemical data on copolymers containing L-alanine or L-phenylalanine

The water-soluble block copolymers, whose syntheses were described in the preceding paper, were subjected to ultracentrifugation, viscosity, optical rotatory dispersion circular dichroism, fluorescence, proton magnetic resonance, and infrared measurements. Discrepancies between M _n and M _w, and line broadening in the proton magnetic resonance spectra (attributed to the formation of rigid structures), support the conclusion (drawn in the preceding paper) that the block copolymers are aggregated in aqueous solution. It is shown that similar block copolymers reported in the literature are also aggregated.     

Conformational studies of polymers and copolymers of L-aspartate ester. II. Infrared studies and the factors involved in the formation of the omega-helix

It has already been show that the helix senses of poly(β-benzyl L -aspartate) and poly(β-methyl L -aspartate) are left-handed, while the poly esters of n-propyl, isopropyl, n-butyl, and phenethyl L -asparate are all right-handed. The effect of changes in helix sense from the left-handed to the right-handed α-helical form on the infrared spectra of copolymers of benzyl L -aspartate with ethyl, n-butyl, isopropyl, n-propyl, and phenethyl L -aspartate have been studied. Those show that for the right-handed helical form the amide band frequencies fall within the range given by Elliott,⁷ while for the left-handed form the frequencies are higher. The frequency ranges for the two helix senses are given and have been used to show that poly (β-n-propyl L -aspartate) in chloroform solution undergoes a transition from the right-handed to the left-handed helix form on heating. Polarized infrared studies of the different copolymers show that the disposition of the side chain ester groups is different for the two forms. Although methyl L -aspartate forms a left-handed α-helix similar to benzyl L -aspartate, the introduction of methyl L -aspartate residues into poly (β-benzyl L -aspartate) prevents the formation of the ω-helix. The factors involved in the formation of this helix form are discussed.     

Block copolymerization of vinyl compounds by the terminal-group activation of poly(α-amino acids) and the characterization of block copolymers

The terminal amino groups of polysarcosine, poly(γ-benzyl l-glutamate), and poly(ε-benzyloxycarbonyl-l-lysine) were haloacetylated. The mixture of the terminally haloacetylated poly(α-amino acid) and styrene or methyl methacrylate was photoirradiated in the presence of Mn₂(CO)₁₀, or heated with Mo(CO)₆, yielding A-B-A-type block copolymers consisting of poly(α-amino acid) (the A component) and vinyl polymer (the B component). The block copolymers were characterized, and the present investigation revealed that the thermally initiated polymerization of vinyl compounds by the trichloroacetyl poly(α-amino acid)/Mo(CO)₆ system was the most suitable for the synthesis of the α-amino acid/vinyl compound block copolymers. The A-B-A type block copolymers showed higher antithrombogenicity than the corresponding homopolymers. In particular, a film of the A-B-A-type block copolymer of poly[Glu(OBzl)] and polystyrene possessed a microphase-separated structure and did not induce a conformational change of fibrinogen adsorbed, leading to a high antithrombogenicity.     

On the amino-acid-sequence distribution in benzylglutamate-methionine copolymers prepared from N-carboxyanhydrides

The amino-acid-sequence distribution in poly(γ-benzyl-L -glutamate, L -methionine) prepared by polymerization of the respective N-carboxyanhydrides has been investigated. This copolymer was converted first to poly(L -glutamic acid, L -methionine), which was subsequently cleaved by treatment with cyanogen bromide. The resulting material was fractionated into oligomers of (glutamic acid)_n-homoserine whose relative molar amounts were determined quantitatively. The results have been compared with those for a random incorporation of the methionine in a γ-benzylglutamate host polymer. Fairly close agreement has been found.     

Synthesis and ordered structure of amphipatic block copolymers with a saccharide and a peptide block

Amphipatic block copolymers (OβEb) with a hydrophilic saccharide block and a hydrophobic polypeptide block were synthesized. In these copolymers the saccharide block is the glyco-amino acid Oβ from ovomucoid and the peptide block (Eb) is a poly(γ-benzyl-L -glutamate) block. Copolymers OβEb exhibit, in the solid state and in Me₂SO concentrated solutions, mesomorphic lamellar structures where the polypeptide chains are in an α-helical conformation. Depending on the molecular weight of the polypeptide block, three types of lamellar structures are obtained, and they differ by the mode of organization of the polypeptide chains in their lamellae and by the T or Y conformation of the saccharide block.     

Nuclear magnetic resonance and optical spectroscopic studies of block copolymers of polypeptides. I. Block copoly((benzyl-L-glutamate) n : (benzyl-L-aspartate) m )

In a study of A–B type block copolymers of γ benzyl-L -gultamate and β-benzyl-L -aspartate use has been made of the observations: (1) that for poly aspartate esters the chemical shifts of the α-CH and NH resonances are sensitive to the helix sense, (2) that in both helical and random coil conformations the same resonances of poly-γ-benzyl-L -glutamate are well separated from those of poly aspartates. Since the sense of poly-β-benzyl-L -aspartate is very sensitive to the inclusion of γ-benzyl-L -glutamate residues, the degree of overlap between the blocks can be studied by monitoring the helix sense of the aspartate. The ability of the NMR method to make separate observation of the two blocks removes the necessity of relying on an overall ORD parameter such as b₀. The copolymers studied include those having lefthanded, righthanded, and mixed-sense aspartate, corresponding to differing degrees of overlap.     

Conformational studies of polymers and copolymers of L-aspartate esters. I. Preparation and solution studies

An alcoholysis method is described for the modification of high molecular weight poly(β-benzyl L -asparatate); by this method the benzyl groups in the polypeptide have been replaced by methyl, ethyl, isopropyl, n-propyl, and phenethyl groups to give a series of copolymers of each of the corresponding aspartate esters with benzyl L -aspartate. By repeating the reactions, replacement of better than 99% has been achieved in some cases to give in effect the homopolymer. Optical rotatory dispersion studies show that of all the systems studied only poly(β-methyl L -aspartate) has the left-handed helix sense, the others are right-handed. It is shown further that the helix sense is not an intrinsic property of the nature of the aspartate side chain. Raising the temperature of chloroform solutions of the right-handed form of the copolymers of benzyl L -aspartate and ethyl L -aspartate results in a transition to the left-handed helix, the temperature of the transition being dependent on the composition of the copolymer. Also poly(β-n-propyl L -aspartate) undergoes a transition from the right- to the left-handed helix form at 59°C. These results suggest a general pattern of behavior of poly(aspartate esters) and that with suitable conditions of solvent and temperature they may be in either the right- or left-handed helical form.     

Comb-shaped graft copolymers with cellulose side-chains prepared via click chemistry

Comb-shaped copolymers with cellobiose acetate or cellulose triacetate (CTA) side-chains, PPMA-g-(CTA2-C15) and PPMA-g-(CTA13-C15), were prepared by grafting N-(15-azidopentadecanoyl)-2,3,6-tri-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-β-d-glucopyranosyl)-β-d-glucopyranosylamine (CTA2-C15-N₃) and N-(15-azidopentadecanoyl)-tri-O-acetyl-β-cellulosylamine (CTA13-C15-N₃, number average degree of polymerization (DP_n) = 13) onto poly(2-propyn-1-yl methacrylate) (PPMA, weight average degree of polymerization (DP_w, X + Y = 5.59 × 10²)) via “click chemistry”. The copolymers were characterized by ¹H, ¹³C and two-dimensional NMR and size exclusion chromatography-multi-angle laser light scattering (SEC-MALS) measurements. The numbers of CTA side-chains (X) of PPMA-g-(CTA2-C15) and PPMA-g-(CTA13-C15) were calculated as 4.03 × 10² and 2.45 × 10², respectively. Copolymers with cellulosic side-chains, PPMA-g-(CELL2-C15) and PPMA-g-(CELL13-C15), were successfully obtained after deacetylation of PPMA-g-(CTA2-C15) and PPMA-g-(CTA13-C15), respectively. X-ray diffraction measurements revealed that PPMA-g-(CELL13-C15) showed crystalline pattern of cellulose II, which is believed to have anti-parallel orientation.     

15N-nmr spectroscopy. 33. Assignments of isotactic and syndiotactic sequences in poly(D,L-amino acids)

¹⁵N-enriched (D ,L -Leu)_n, (γ-OMe-D ,L -Glu)_n, (D ,L -Val)_n, and (D ,L -Phe)_n were prepared, 40.55-MHz ¹⁵N-nmr spectra were measured in various solvents. The signal patterns depend strongly on the nature of the solvent, yet in most cases at least four signals are resolved, representing the four enantiomeric pairs of triads L -L -L (D -D -D ), L -D -L (D -L -D ), L -L -D (D -D -L ), and D -L -L (L -D -D ). Numerous copolypeptides of the general structure (A)_n-B*-(A)_m (the asterisk denotes 40–50% ¹⁵N enrichment) were synthesized and measured as models for syndiotactic sequences in the spectra of poly(D ,L -amino acids). In this way unambiguous assignments for both isotactic and syndiotactic trials were obtained. A spectroscopic rule was established: “isotactic sequences absorb downfield of syndiotactic ones.” Furthermore, the spectra of various types of stereocopolypeptides such as (L -Leu/L -Val)_n and (L -Leu/D -Val)_n were investigated, including the ternary systems (L -Leu/L -Ala/D -Ala)_n (L -Leu/L -Ala/Gly)_n, (L -Leu/D -Ala/Gly)_n, (L -Val/L -Ala/Gly)_n, and (L -Val/D -Ala/Gly)_n. All copolymerization of D - and L -amino acid NCAs investigated in this work showed a low degree of stereoselectivity.     

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.     

The random coil leads to beta transition of copolymers of L-lysine and L-valine: potentiometric titration and circular dichroism studies.

A series of copolymers of L -lysine and L -valine [poly(L -lysine^f L -valine^100-f)] containing 0–13% L -valine have been studied, in 0.10M KF solution, using potentiometric titration and circular dichroism spectroscopy. Incorporation of increasing amounts of valine into the copolymers favors β-sheet formation over α-helix formation at high pH and room temperature. The titrations were analyzed using the method of Zimm and Rice and the partial free energy (ΔG⁰_cβ) for the coil-to-β-sheet transition for valine is estimated at 900 cal/mole at 25°C. From the temperature dependence of the free energy, the partial enthalpy, ΔH⁰_cβ, and entropy, ΔS⁰_cβ, of the transition for valine is estimated to be 854 cal/mole and 6.0 e.u., respectively. The corresponding partial thermodynamic parameters for L -lysine are in agreement with published results. The fraction of β-sheet versus pH has been calculated for poly(L -lysine^86.8 L -valine^13.2) at 25.0°C using the titration data; data obtained from circular dichroism spectroscopy for the same copolymer are in good accord. It is concluded from these results that L -valine is a very strong β-sheet forming amino acid. Furthermore, these results indicate that the Zimm–Rice method is applicable to transitions between the coil and β-sheet states for a polypeptide containing two different residues.     

Specificity of DNA-basic polypeptide interactions. III. Sequential and random copolymers of lysine and aromatic amino acids.

Sequential and random lysine copolymers containing various amounts of different aromatic amino acids were synthesized. The sequential copolypeptides exhibited strong dependence of yield and degree of polymerization on the amino acid sequence of the repeating unit. To elucidate the specific contributions of aromatic side chains to the interaction of these copolymers with DNA, direct-mixed complexes were studied by thermal denaturation and CD. The melting behaviour of peptide-bound DNA was found to be strongly affected by amino acid composition and sequence. The contribution of the different aromatic amino acids to thermal stability decreased in the order: polylysine > [Lys, Tyr]_n > [Lys,Phe]_n > [Lys,(OMe)Tyr]_n. The CD spectrum of DNA was altered by random copolymers, whereas sequential copolymers exhibited no changes. The influence of the random copolymers on the CD spectrum of DNA decreased in the series: polylysine > [Lys,Phe]_n > [Lys,(OMe)Tyr]_n > [Lys,Tyr]_n. The contribution of the different aromatic amino acids to thermal stability is interpreted as stacking tendencies toward denatured and, in the case of Tyr, H-bond formation with native DNA. The differences found for the random and the sequential polypeptides can best be explained by assuming a cooperative action of rather small peptide segments.     

Molecular mobility of polypeptides containing proline as determined by 13C magnetic resonance

The molecular conformations and dynamics of poly(L -prolyl), poly(hydroxyl-L -prolyl), poly(L -prolyl-glycyl), poly(hydroxyl-L -prolyl), and poly(glycyl-glycyl-L -prolyl-glycyl), in aqueous solution, have been studied using ¹³C pulse Fourier transform nmr spectroscopy. From a measurement of the intensities of major and minor resonances in the spectra of the copolypeptides, it was determined that 15–20% of the glycyl-prolyl and glycyl-hydroxyprolyl peptide bonds are cis. Effective rotational correlation times (τ_eff), obtained from measurements of spin-lattice relaxation times (T₁) of individual backbone and side-chain carbons, demonstrated that backbone reorientation is approximately isotropic for the five polypeptides and is characterized by correlation times of ca. 0.3–0.6 nanoseconds as a result of rapid segmental motion. In a given polypeptide glycyl and pyrrolidine residues were found to have the same backbone correlation times, but backbone carbon τ_eff values did decrease as the glycyl content of the peptides increased. A semi-quantitative analysis of C^β, C^γ, and C^δ correlation times suggests that rapid ring motion in both prolyl and hydroxyprolyl involves primarily C^γ and C^β, with the prolyl ring being more mobile than the hydroxyprolyl ring.     

Basic polypeptides as histone models. Synthesis, conformation, and interaction with DNA of statistical copolymers (Lys x,Ala y)n.

Statistical copolymers (Lys^x,Ala^y)_n were synthesized by copolymerization of N-carboxyanhydrides of L -amino acids. The conformation of copolymers in aqueous solutions was investigated using circular dichroism (CD). Calculations based on the CD data showed that polymers (Lys^x,Ala^y)_n can exhibit a random conformation, an α-helix, and a β-structure in various ratios. CD spectra of complexes of copolymers with DNA prepared by gradual dialysis from a high ionic strength to 0.15 M NaCl can be correlated with the copolymer conformation in medium and high ionic strength. For copolymers forming an α-helix and β-structure, these spectra show resemblance with similar spectra of complexes of those histones that are able to exhibit ordered conformations.     

Conformational induction in copolypeptides of the form AnBm

The polymerization kinetics and optical rotatory properties of A_nB_m polypeptides have been studied, where A = D ,L -Tyr, D ,L -TyrZ, D ,L -LysZ, L -AspOBzl, or L -Asp-ONBzl, and B = D ,L -GluOR (R = Me or Bzl). In most cases where A_n and B_m prefer the same helix sense, the polymerization of A N-carboxyanhydride (initiated by B_m in dioxane) shows first order kinetics and produces a monotonic change in optical rotation, while if opposite helix senses are preferred, the kinetics are multiphasic and the change in rotation reverses direction after the addition of several residues. The rotation change in the latter case is interpreted to mean that the helix in the A block is initially induced to take the nonpreferred sense, as originally suggested by Doty and Lundberg from similar observations on (D -GluOBzl)_n-(L -GluOBzl)_m. It is found here that the CD spectra for the latter polymer show the sign changes required by this hypothesis. The optical rotation curves and CD spectra for (D ,L -Tyr)_n-(L -GluOBzl)₂₀ suggest, by analogy, that (L -Tyr)_n prefers the same helix sense as (L -GluOBzl)_n. However, it is found that the opposite conclusion is equally consistent with the data if one considers the effects of possible changes in side-chain conformation on these data in accordance with the calculated CD spectra of Chen and Woody. The optical rotation curves for (D -GluOBzl)_n-(L -GluOBzl)₂₀, (D -Tyr)_n-(L-GluOBzl)₂₀, and (L -Tyr)_n-(L -GluOBzl)₂₀ are all found to be consistent with a two-state equilibrium model in which the A block initially takes on an induced conformation and has an increasing tendency to revert to its preferred conformation as n increases. It is concluded that in both D -Tyr and L-Tyr the side-chain and/or the backbone conformation is induced by the neighboring L -GluOBzl block, and the data do not distinguish which type of change is occurring. These results are discussed in connection with other observations bearing on the helix sense of (L -Tyr)_n.     

13C Nuclear magnetic resonance study of salt induced conformational change of basic polypeptides: Poly (L-lysine), poly (L-arginine), and poly (L-ornithine)

A ¹³C-nmr study of the salt-induced helix–coil transition of the basic polypeptides poly(L -lysine) [(Lys)_n], poly(L -arginine) [(Arg)_n], and poly (L -ornithine) [(Orn)_n] was performed to serve as a reference of the helical portion of histones and other proteins. As is the case with pH-induced helix–coil transition, the downfield displacement of the C^α and carbonyl carbon signals are observed in the helical state. The upfield shift of the C^β signals, on the other hand, is noted in the salt-induced transition. Regardless of the differences in the side chains and also the salts used, very similar helix-induced chemical shifts are obtained for (Lys)_n and (Arg)_n. However, the displacement of the C^α, C^β, and carbonyl carbons of (Orn)n in the presence of 4M NaClO₄ is found to be almost 50% of that of (Lys)_n and (Arg)_n. This is explained by the fact that the maximum helical content is about 50%, consistent with the ORD result. Further, the motion of the backbone and side chains of the helical from was estimated by measuring the spin-lattice relaxation time (T₁), nuclear Overhauser enhancement (NOE), and line width. In the case of (Lys)_n, the motion of the side chains is charged very little in comparison with that of the random coil. Indicating that the aggregation of the salt-induced helix is small in contrast to that of the pH-induced helix. For (Arg)_n, however, the precipitate of the helical polymers is mainly due to aggregation.     

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.     

15N nmr spectroscopy. I. Polysarcosine and related sequence polypeptides

The natural abundance ¹⁵N nmr spectra of linear polysarcosine (DP = 35) has been recorded in Me₂SO and H₂O solution. Because of cis/trans isomerization at the peptide bond, a broad signal with several splittings was observed. These splittings appear to reflect the influence of three peptide bonds on a single N atom. The ¹⁵N signals from the sequence polypeptides (β-Ala-Sar-Gly)_n and (β-Ala-Sar-D ,L -Ala)_n also show a cis/trans splitting, as well as chemical shifts which are dependent on the peptide sequence. The tertiary nitrogen of the sarcosyl residue has a T₁ relaxation time which is longer than the T₁ for secondary nitrogens of the other amino acids. The nuclear Overhauser effect is also discussed.     

Block oligopeptides (L-lysyl)m-(L-alanyl)n-L-Tyrosyl-(L-Alanyl)n-(L-Lysyl)m. II. Circular dichroism and pulsefluorimetry conformational studies

The conformation of chromatographically pure block oligopeptides (L -lysyl)_m-(L -alanyl)_n- L -tyrosyl-(L -alanyl)_n-(L -lysyl)_m with n = 3 and m = 6 or 3 is investigated. By circular dichroism it is shown that these peptides may exhibit a partially α-helical structure depending upon pH, ionic strength, solvent, and temprerature. An attempt is made to describe the helical content of these small peptides by utilizing the data obtained on high-molecular-weight poly(L -lysine). By measurement of the quantum yield and the decays of the peptides fluorescence, it is shown that, in aqueous solution, at neutral pH, the fluorescence of the peptides is quenched by interactions with the peptide carbonyl groups. The decays are multiexponential, which shows the presence of several conformations of the phenolic chromophore relative to the peptide chain. The addition of methanol, which induced the helix formation, decreases the quenching of the fluorescence and the multiexponential character of the decays. In presence of sodium hydroxide, which further increases the helical content of the peptides, a dynamic quenching occured that can be attributed to interactions between the phenol hydroxyl group of tyrosine (ith residue) and the ε-amino groups of the (i+4)th and (i -4)th lysyl residues.