Occurrence of beta-chain conformation in poly-gamma-methyl glutamate membranes

Molecular chain conformations of poly-γ-methyl-L -glutamate, poly-γ-methyl-D -glutamate, and poly-γ-methyl-D ,L -glutamate in membranes prepared by using mainly trifluoroacetic acid and formic acid as solvents were investigated by infrared, X-ray diffraction, and optical rotatory dispersion measurements. It was pointed that these polymers exist in the α-helix form in membranes cast from trifluoroacetic acid solutions, but in the β-chain form in membrances swollen in formic acid. The β-chain structure was also observed in crystals precipitated from dilute solutions including formic acid. The formation of the β-chain structure was discussed.     

Spectroscopic studies of random chain and -helical polypeptides in hexafluoroisopropanol

The infrared, ultraviolet, circular dichroism, and optical rotatory dispersion spectra of five synthetic polypeptides dissolved in hexafluoroisopropanol are reported. This solvent is useful because it dissolves most proteins and non-ionic polypeptides and also is transparent in spectral regions critical for polypeptide conformational diagnoses. Poly-γ-morpholinylethyl-L -glutamamide has random chain type spectra in this solvent, whereas the spectra of poly-γ-methyl-L -glutamate, poly-L -methionine, poly-ε, N -Carbo-benzoxy-L -lysine, and poly-L -homoserine indicate that these four polypeptides are α-helical. Small but significant variability between the different α-helical polypeptides is seen in their circular dichroism spectra and optical rotatory dispersions. An argument is presented that these differences may be due to slight geometry differences between different α-helices.     

Rotatory dispersion and circular dichroism of low-molecular-weight poly- -benzyl-L-glutamate

Rotatory dispersion and circular dichroism of low-molecular-weight poly-γ-benzyl-L -glutamate, which was prepared from the N-carboxyanhydride by n-hexylamine initiation at [A]/[I] 3, 4, and 8, have been measured in ethylene dichloride and dioxane at different concentrations. The rotatory properties of the polypeptides are all characterized by a trough at 233 mμ of a negative Cotton effect or by a negative circular dichroic band at 223 mμ. With increasing A/I value or concentration, dextrorotation increases and the negative dichroic band becomes deeper. Both the trough magnitude and the negative ellipticity are linearly dependent on the content of β-structure, and the rotatory parameters for the pure β-structure are estimated by extrapolation of the linear relations. Circular dichroism and infrared spectra of the polypeptides have also been measured in trifluoroethanol, and the effect of solvents on the polypeptide conformation is discussed.     

Syntheses and conformational studies of poly- ,N-alkyl L-asparagines

Several β,N-alkyl L -asparagines were prepared from the phthalyl and benzyloxycarbonyl derivatives. High-molecular-weight poly-β,N-benzyl L -asparagine and poly-β,N-(1)-phenethyl L -asparagine were prepared from the corresponding N-carboxyanhy-drides. From the results obtained by a study of the infrared absorption spectra and the optical rotatory dispersion, poly-β-N-benzyl L -asparagine was found to be a random coil structure in dichloroacetic acid and the optical rotatory dispersion curves gradually changed into the left-handed α-helix structure when chloroform was added to the solution. The coil-to-helix transition was observed in the vicinity of 20% dichloroacetic acid in a dichloroacetic acid-chloroform mixture. Poly-β,N-(d), (l), and (d + l, 1:1)

1 (d + l, 1:1): mixed polymer containing the same weighed poly-β,N-(d) and (l)-(1)-phenethyl L -asparagines.

-(1)-phenethyl L -asparagines showed a nearly constant specific rotation in the dichloroacetic acidchloroform solvent system. Poly-β,N-(dl)-(1)-phenethyl L -asparagine caused a gradual folding of the helix at dichloroacetic acid content of less than 20%.     

Thermal and charge-induced coil to -helix transition of poly-L-glutamic acid and random L-glutamic acid-L-alanine copolymers

Thermal and charge induced random coil to α-helix transitions of poly-L -glutamic acid (PGA) were measured by optical rotatory dispersion in various solvents. The data of PGA in 0.1M Nacl were analyzed by the Zimm-Rice theory. Enthalpy and entropy changes for the coil-to-helix transition in the unionized state were obtained: ΔH° = ?1020 ± 100 cal/residue mole; ΔH° = ?3.0 ± 0.4 e.u./residue mole. The initiation parameter, σ, of the Zimm-Rice theory was given by a value of 5 ± 1 × 10^-3. Random copolymers of L -glutamic acid and L -alanine containing 10, 30, and 40 molar percents of alanyl residue were synthesized. Stabilities of α-helix of the copolymers were compared to that of PGA. In water and water-ethanol solutions, stabilities of the polymers were almost equal after the simple correction about the ionized charge density of the polymers. In 0.1 M NaCl solution these copolymers showed some deviations from the transition curve of PGA, which would suggest the hydrophobic contribution of the alanyl residues.     

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.     

Conformational studies of anthrax polypeptide, subtilis polypeptide, and synthetic poly- -L-glutamic acid

Poly-γ-L -glutamic acid has been synthesized by the activated pentachlorophenyl ester polymerization method and the molecule weight of the polymer was found to be 16,000. Comparative conformational studies on the synthetic and on the native polyglutamic acid mbtained from B. anthracis and B. subtilis were carried out using optical rotatory dispersion, circular dichroism, peptide absorption spectrum, and titration data. These results show that poly-γ-glutamic acid does not exhibit any conformational order under the conditions of investigation. At low degrees of ionization, restriction of conformational freedom via random “hypercoiling” of the chain appears likely.     

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.     

Structure of aggregates and helix-coil transition of polypeptides by dielectric measurements. I. Poly-gamma-benzyl-L-glutamate in dioxane and dioxane-dichloroacetic acid

Dielectric dispersion measurements on poly-γ-benzyl-L -glutamate (PBLG) in dioxane and dioxane–dichloroacetic acid (DCA) mixtures in the frequency range 200 Hz–2 MHz were made in order to study the structure of molecular aggregates. The structure of aggregates is explained on the basis of the variation of dipole moment and relaxation time with degree of aggregation. PBLG was found to form linear head-to-tail-type aggregates in dioxane. These aggregates gradually reduce in size without loosing their α-helical structure during the process of disaggregation obtained by either adding DCA to the solution in dioxane or by heating. It was confirmed that the addition of 30 wt % DCA completely destroys the aggregation of PBLG in dioxane at 30°C. Thermal disaggregation, however, was not complete even at a temperature approaching the boiling point of the solvent. A reaction scheme for aggregation is proposed and equilibrium constants are calculated at various stages of aggregation. The enthalpy of aggregate formation is found to be ?3 kcal/mol. Results of optical rotatory dispersion measurements of the helix–coil transition in this system are also presented.     

Computer analyses of characteristic infrared bands of globular proteins.

Infrared spectra of myoglobin, ribonuclease, lysozyme, α-chymotrypsin, α-lactalbumin, and β-lactoglobulin A were obtained in deuterium oxide solution in units of absorbance versus wavenumber from 1340 to 1750 cm^?1. The spectra were resolved into Gaussian components by means of an iterative computer program. Resolved characteristic absorption peaks for the two infrared active amide I′ components of antiparallel chain-pleated sheets (β-structure) were obtained. The characteristic amide I′ peaks of α-helical regions and apparently unordered regions overlap in D₂O solution. Absorptivity values for the resolved β-structure peak around 1630 cm^?1 were estimated on the basis of the known structure of ribonuclease, lysozyme, and β-chymotrypsin. The β-structure content of β-lactoglobulin was estimated to be ca. 48% of α-lactalbumin ca. 18%, and of α_s-casein close to zero. The results are in general agreement with conclusions drawn from circular dichroism and optical rotatory dispersion studies.     

Helix-coil transition of poly-gamma-N-carbobenzoxy-L-alpha, gamma-diaminobutyrate and poly-delta-N-carbobenzoxy-L-ornithine

The helix–coil transition of poly-γ-N-carbobenzoxy-L -α,γ-diaminobutyrate (PCLB) and poly-δ-N-carbobenzoxy-L -ornithine (PCLO) in chloroform–dichloroacetic acid mixtures was followed by optical rotatory dispersion. PCLB displays a “normal” temperature-induced transition, but PCLO an “inverse” one. The thermodynamic parameters for helix formation of the two polymers were determined using the Zimm-Bragg theory. The enthalpy for adding an amide residue to a helical region, ΔH, and the initiation factor σ were ΔH = ?180 cal/mole and σ = 9.2 × 10^?5 for PCLB and ΔH = +490 cal/mole and σ = 1.9 × 10^?5 for PCLO.     

Rotatory dispersion of the β-form of polypeptides

The rotatory dispersion of the β-form of polypeptides is considered. The parameter b₀ of the Moffit-Yang equation is calculated quantum mechanically. The results obtained b₀ = ?30, agrees with experiment which gives b₀ near zero for the β-form.     

Optical activity of polypeptides and proteins

Using methods described in a previous publication the optical activity of a number of polypeptides and proteins has been calculated. The systems included the α-helix, the two β-structures, polyproline I, polyproline II, collagen and collagen models, and poly-N-methylalanine. In addition to these orderded structures, calculations were also performed on the α, β and nonperiodic regions of myoglobin, lysozyme, ribonuclease-S and β-chymotrypsin. The α and β structures in prteins differ from the polypeptide models by being very short and partially disordered. It is concluded that the 222-nm band of the α-helix is a good method for detecting helices in proteins but that the 207-and 191-nm bands of the helix will not fit a linear superposition model. The circular dichroism of the so-called β regions of proteins differs markedly from that for ideal β structure because of breakdownin symmetry. As a result estimates of β-structure in proteins based on polypeptide models are not likely to be quantitative. The theoretical methods give an adequate account of the optical activity of all the ordered polypeptides except polyproline II and collagen and (by inference) the nonperiodic chains in the various proteins. This difficulty is the remaining barrier to a complete theory of the optical activity of the polypeptide backbone in globular proteins.     

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     

Structure of Bovine αs-Casein

The secondary structure of bovine α_s-casein and chemically modified α_s-casein in various solvents was investigated by infrared absorption spectrum and optical rotatory dispersion measurements. Amino groups of α_s-casein were either succinylated or acetylated, and carboxyl groups were either methylated or ethylated. Acetylated- and ethylated-α_s-caseins are insoluble in water. Water-soluble samples have unordered structure in water. In organic solvents, such as 2-chloroethanol and ethylene glycol, they have about 50% α-helical fraction. On the other hand, it was found that methylated-α_s-casein had two infrared absorption peaks centered at 1625 and 1643 cm^?1 in D₂O-CH₃OD mixed solvent. This fact may be connected with the presence of β-structure. In the case of solid film of this sample, cast from solution containing CH₃OH, the presence of β-structure was indicated, too. The authors attempted to explain the formation of β-structure in methylated-α_s-casein in terms of the electrostatic interactions due to the differences in the net charge between methylated and unmodified α_s-caseins.     

Conformation of poly-L-tyrosine in trimethyl phosphate solution

Absorption, circular dichroism (CD), and optical rotatory dispersion (ORD) measurements were carried out on poly-L -tyrosine in trimethyl phosphate solution over the spectral range 185–600 mμ. There is evidence in the CD spectrum for side chain-side chain interactions in poly-L -tyrosine. ORD and CD data in dimethylformamide and pyridine closely parallel those in trimethyl phosphate, indicating a similarity in conformation of the polymer in all three solvents. In the polarized infrared spectrum both position and polarization of amide A, I, and II bands are characteristic of α-helical polypeptides. Bands corresponding to side chain also exhibit dichroism, suggesting that the side chains are not randomly oriented. Viscosity and light-scattering studies are consistent with α-helical structure for the polymer that, remains rigid over a temperature range of 15–50°C and becomes somewhat flexible at higher temperatures. Optical rotatory properties were found to vary gradually and continuously with temperature over the range of ?30 to +100°C. This suggested that all three electronic transitions of tyrosyl side chain are optically active, and that the side chains have some freedom of motion that decreases with decreasing temperature, disappearing only at about ?30°C.     

Optical studies on the structure of hemoglobin messenger ribonucleic acid

The secondary structure of hemoglobin mRNA (HbmRNA has been investigated by optical rotatory dispersion (ORD), circular dichroism (CD) and ultraviolet absorbance spectrophotometry. The dependence on temperature or reaction with formaldehyde of the CD and absorbance are characteristic of a structure with substantial base pairings at 20°C. The presence of Cotton effects in one of the major ultraviolet absorption bands indicates a highly ordered secondary structure. The UV hyperchromism on thermal denaturation is consistent with a value of 58% double helical content.     

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.     

Raman spectroscopic study of poly (β-benzyl-L-aspartate) and sequential polypeptides

Poly-β-benzyl-L -aspartate (poly[Asp(OBzl)]) forms either a lefthanded α-helix, β-sheet, ω-helix, or random coil under appropriate conditions. In this paper the Raman spectra of the above poly[Asp(OBzl)] conformations are compared. The Raman active amide I line shifts from 1663 cm^?1 to 1679 cm^?1 upon thermal conversion of poly[Asp(OBzl)] from the α-helical to β-sheet conformation while an intense line appearing at 890 cm^?1 in the spectrum of the α-helix decreases in intensity. The 890 cm^?1 line also displays weak intensity when the polymer is dissolved in chloroform–dichloroacetic acid solution and therefore is converted to the random coil. This line probably arises from a skeletal vibration and is expected to be conformationally sensitive. Similar behavior in the intensity of skeletal vibrations is discussed for other polypeptides undergoing conformational transitions. The Raman spectra of two cross-β-sheet copolypeptides, poly(Ala-Gly) and poly(Ser-Gly), are examined. These sequential polypeptides are model compounds for the crystalline regions of Bombyx mori silk fibroin which forms an extensive β-sheet structure. The amide I, III, and skeletal vibrations appeared in the Raman spectra of these polypeptides at the frequencies and intensities associated with β-sheet homopolypeptides. Since the sequential copolypeptides are intermediate in complexity between the homopolypeptides and the proteins, these results indicate that Raman structure–frequency correlations obtained from homopolypeptide studies can now be applied to protein spectra with greater confidence. The perturbation scheme developed by Krimm and Abe for explaining the frequency splitting of the amide I vibrations in β-sheet polyglycine is applied to poly(L -valine), poly-(Ala-Gly), poly(Ser-Gly), and poly[Asp(OBzl)]. The value of the “unperturbed” frequency, V₀, for poly[Asp(OBzl)] was significantly greater than the corresponding values for the other polypeptides. A structural origin for this difference may be displacement of adjacent hydrogen-bonded chains relative to the standard β-sheet conformation.     

Secondary Structure of Nuclease P1

The molecular conformation of nuclease P₁ in aqueous solution was investigated by measuring the optical rotatory dispersion (ORD) and circular dichroism (CD). The optical rotatory dispersion constant, λ was 281 nm. The Moffit-Yang parameters, a₀ and b₀, were ?2 and ?195, respectively. The ORD spectrum showed a minimum at 234 nm and the reduced mean residue rotation at 233 nm, [m]233, was ?5880. The CD spectrum showed a double minimum at 213 and 226 nm and the molecular ellipticity at 222 nm, [θ]22, was -11,900. From these data, the α-helix content was calculated to be 29 to 31 %. The computer fit of CD suggests that the α-structure is about 6% and the random coil is about 63%. The helical structure was found to be quite stable to denaturing reagents such as urea and guanidine hydrochloride. However, removal of zinc atoms from the enzyme resulted in disruption of the helical structure with inactivation.