Raman spectra of D and L amino acid copolymers. Poly-DL-alanine, poly-DL-leucine, and poly-DL-lysine.

The Raman spectrum of poly-DL -alanine (PDLA) in the solid state is interpreted in terms of the disordered chain conformation, in analogy with the spectrum of mechanically deformed poly-L -alanine. The polymer is largely disordered with only a small α-helical content in the solid state. When PDLA is dissolved in water, the spectra suggest that short α-helical segments are formed upon dissolution. These helical regions might be stabilized by hydrophobic bonds between side-chain methyl groups. Addition of methanol to the aqueous PDLA solutions results in a Raman spectrum resembling that of solid PDLA. This result suggests that the methanol disrupts the helical regions by breaking the hydrophobic bonds. The Raman spectra of poly-DL -leucine (PDLL) and poly-L -leucine (PLL) are compared and only slight differences are observed in the amide I and III regions, indicating that PDLL does not have an appreciable disordered chain content. Significant differences are observed in the skeletal regions. The 931-cm^?1 lines in the PLL and PDLL spectra are assigned to residues in α-helical segments of the preferred screw sense, i.e., L -residues in right-handed segments and D -residues in left-handed segments (in PDLL). On the other hand, the 890-cm^?1 line in the spectrum of PDLL is assigned to residues not in the preferred helical sence, i.e., L -residues in left-handed segments and D -residues in right-handed ones. The Raman spectra of poly-DL -lysine and poly-L -lysine in salt-free water at pH 7.0 are compared. The Raman spectra of the two polymers are very similar. However, this does not negate the hypothesis of local order in poly-L -lysine because the distribution of the residues in poly-DL -lysine probably tends towards blocks, and the individual blocks may take up the 3₁ helix.     

Circular dichroism and proton magnetic resonance studies of random chain poly-L-lysine

The 218-nm peak, characteristic of the circular dichroism of randomly coiled poly-α-amino acids can be demonstrated in solutions of penta-L -lysine, α-glycyl-L -lysine, as well as poly-L -lysine. The thermal stability of the particular state that gives rise to this 218-nm band in the CD is similar for all three peptides. These results eliminate the possibility that poly-L -lysine forms a structure with long-range order in acidic aqueous solution since the stability of such a structure would be expected to be greater for a higher molecular weight polymer than for a pentamer. The intrinsic viscosity of poly-L -lysine of molecular weight 180,000 varies only slightly between 25 and 60°C. The proton magnetic resonance spectra of poly-L -lysine and penta-L -lysine are indistinguishable on the basis of the chemical shift of all resonances, their line widths, and the exchange rates of the N? H protons. This demonstrates that poly-L -lysine does not possess a cooperatively formed ordered structure in acidic solutions. A weak band at 238 nm is observed in the circular dichroism of poly-L -lysine and other peptides. It is suggested that the effects of change in temperature, salt concentration, or polymer on both the magnitude and position of the 238-nm band may be explained if it is assumed that it is a shoulder of a lower wavelength peak.     

Interaction of poly-L-lysine with nucleic acids. II. Poly(A+U), poly(A+2U), and rice dwarf virus RNA

The optical density–temperature profile of double-stranded poly(A + U), triple stranded poly(A + 2U), and double-stranded RNA from rice dwarf virus in solutions with and without poly-L -lysine has been examined. When poly-L -lysine is added, more than one melting temperature T_m is observed for poly(A + U) and poly(A + 2U). One of them is considered to correspond to the melting of the polynucleotide molecule free from poly-L -lysine, and another to the melting of a polynucleotide–poly-L -lysine complex. For rice dwarf virus RNA, the T_m assignable to the complex is not found to be lower than 99°C. In every case, however, the hyperchromicity observed at the T_m of the free poly-nucleotide molecule is lowered linearly as the amount of poly-L -lysine added to the solution increases. This fact is taken as indicating that there is a stoichiometric complex formed. The stoichiometric ratio lysine/nucleotide in each complex is determined by examining the relation between the amount of poly-L -lysine added to the solution and the percentage of hyperchromicity remaining at T_m of the free polynucleotide molecule. The ratio is found to be 2/3 for all of the three complexes. A discussion is given on the molecular conformations of four types of polynucleotide–polylysine complex hitherto found: (A) double-stranded DNA plus poly-L -lysine in which the lyslne/nucleotide ratio is 1, (B) three-stranded RNA [poly(A + 2U)] plus poly-L -lysine in which the ratio is 2/3, (C) double-stranded RNA [poly (A + U) or rice dwarf virus RNA] plus poly-L -lysine in which the ratio is 2/3, and (D) double-stranded RNA [poly(I + C)] plus poly-L -lysine in which the ratio is 1/2.     

Interaction between chondroitin-6-sulfate and poly-L-arginine in aqueous solution

The interactions between chondroitin-6-sulfate and poly-L -arginine in aqueous salt solution have been investigated by circular dichroism techniques. In the presence of chondroitin-6-sulfate, at neutral pH, poly-L -arginine adopts the α-helical conformation rather than “charged coil” form observed in the absence of mucopolysaccharide. This interaction is at a maximum when the ratio of arginine to disaccharide residues is 2:1. Elevation of the temperature leads to a sharp melting transition at 76.0 ± 1.0°C. This behavior is in marked contrast to that for poly-L -lysine-chondroitin-6-sulfate interactions, which are at a maximum at a 1:1 residue ratio and have a melting transition at 47.0 ± 1.0°C. These results indicate a stronger interaction for poly-L -arginine than for poly-L -lysine. The positive arginine side chains appear to interact with both the negative sulfate and carboxyl residues, while those of the lysines are involved only with the sulfates. Poly-L -ornithine at neutral pH shows no conformation directing interaction with chondroitin-6-sulfate, although a small proportion of α-helix is formed on dilution of the mixture with methanol. The extent of the interaction of cationic polypeptides with chondroitin-6-sulfate increases in the order poly-L -ornithine, poly-L -lysine, poly-L -arginine, i.e., in the order of increasing side-chain length.     

Calorimetric measurement of enthalpy change in the isothermal helix--coil transition of poly-L-lysine in aqueous solution

The heat ΔH° for converting an uncharged lysine residue from a coil to an α-helical state in poly-L -lysine in 0.1N KCl has been determined calorimetrically to be ?1200 cal/mole at both 15°C and 25°C. Essentially the same value has been obtained for the conversion of an uncharged residue from a coil to a β-pleated sheet state. Titration data provided information about the state of charge of the polymer in the calorimetric experiments, and optical rotatory dispersion data about its conformation. In order to compute ΔH°, the observed Calorimetric heat was corrected for the heat of breaking the sample cell, the heal of dilution of HCl, the heat of neutralization of OH^? ion, and the heat of ionization of the ε-amino group in the random coil. The latter was obtained from similar Calorimetric measurements on poly-D ,L -lysine, which was shown to be a good model for the random coil form of poly-L -lysine. The measured transition heat was ～0.7 cal., which is only 7% of the total heat liberated when a 40 ml solution of 0.25% w/v poly-L -lysine is brought, from pH 11 to pH 7; nevertheless it could be determined with a precision of ±8%. The conformation of poly-L -lysine at pH 11 appears to be completely helical at 15°C, but a mixture of 90% α-helix, 5% β form, and 5% coil at 25°C. Since ΔH° ～ 0 for the α ? β conversion, the polymer behaves like one of 95% α-helix and 5% coil in the calorimeter at 25°C. At neutral pH, poly-L -lysine is an extended coil, like poly-D ,L -lysine.     

Interactions between chondroitin-6-sulfate and poly-L-lysine in aqueous solution: Circular dichroism studies

The interactions between chondroitin-6-sulfate (chondroitin sulfate C) and poly-L -lysine have been studied as models for investigation of possible complex formation between fibrous proteins and mucopolysaccharides. Results obtained using circular dichroism spectroscopy show that poly-L -lysine adopts the α-helical conformation in dilute aqueous salt solution at pH 7 when mixed with chondroitin-6-sulfate, rather than the “charged-coil” observed in the absence of this mucopolysaccharide. This conformation-directing interaction is at a maximum when the ratio of lysine to disaccharide residues is 1 : 1. Changes in the CD spectrum of a 1 : 1 mixture following increase in the salt concentration, or addition of non-polar solvents, indicate that the interaction is ionic in nature. No such effects are observed for non-sulfated mucopolysaccharides mixed with poly-L -lysine, suggesting that, for chondroitin-6-sulfate, it is the sulfate groups rather than the carboxyls which interact with the amine groups of the polypeptide. Elevation of the temperature leads to disruption of the interactions between the polypeptide and polysaccharide species. A sharp melting transition occurs at 47.0 ± 1.0°C, when the poly-L -lysine reverts to the “charged-coil” conformation. The sharp transition suggests that regular ionic bonds are formed between the polypeptide and polysaccharide. These results suggest that a conformation-directing interaction may occur between sulfated mucopolysaccharides and the polar regions of collagen and other fibrous proteins.     

Lysine oligopeptides. Preparation by ion-exchange chromatography

The preparation of L -lysine peptides (Lys_n, n = 2–14) from polyL -lysine is described. Fractionation by ion-exchange column chromatography of poly-L -lysine hydrolysates on a preparative scale resulted in 0.2–1.0 g quantities of individual members of the poly-L -lysine series. The peptides isolated proved to be analytically pure and the optical configuration was fully retained, as demonstrated by complete enzymic digestion. Peptides higher than n = 14 were also prepared. They consisted of oligolysine groups of narrow and accurately determined size distribution. Potentiometric titrations were used both to characterize the products and to demonstrate the characteristic dependence of the dissociation constants on size of the peptide.     

Hydrolysis of poly-L-lysine by pronase

Pronase E is able to hydrolyze poly-L -lysine in 100% yield into the monomer L -lysine. The experiments show an intermediate production of tri-L -lysine and di-L -lysine that are clipped off from the polymeric peptide. During the hydrolytic process a number of characteristic amino acids are released from the enzyme that may indicate its activation. Amino acids as well as mono-L -lysine are decomposed by pronase E after a period of several days.     

Flow-induced conformational changes and phase behavior of aqueous poly-L-lysine solutions

Poly-L -lysine exists as an α-helix at high pH and a random coil at neutral pH. When the α-helix is heated above 27°C, the macromolecule undergoes a conformational transition to a β-sheet. In this study, the stability of the secondary structure of poly-L -lysine in solutions subjected to shear flow, at temperatures below the α-helix to β-sheet transition temperature, were examined using Raman spectroscopy and CD. Solutions initially in the α-helical state showed time-dependent increases in viscosity with shearing, rising as much as an order of magnitude. Visual observation and turbidity measurements showed the formation of a gel-like phase under flow. Laser Raman measurements demonstrated the presence of small amounts of β-sheet structure evidenced by the amide I band at 1666 cm⁻¹. CD measurements indicated that solutions of predominantly α-helical conformation at 20°C transformed into 85% α-helix and 15% β-sheet after being sheared for 20 min. However, on continued shearing the content of β-sheet conformation decreased. The observed phenomena were explained in terms of a “zipping-up” molecular model based on flow enhanced hydrophobic interactions similar to that observed in gel-forming flexible polymers. © 1998 John Wiley & Sons, Inc. Biopoly 45: 239–246, 1998     

Raman spectroscopic study of tropomyosin denaturation

Raman spectra of the pH denaturation of tropomyosin are presented. In the native state tropomyosin has an alpha-helical content of nearly 90%, but this value drops rapidly as the pH is raised above 9.5. The Raman spectrum of the native state is characterized by a strong amide I line appearing at 1655 cm^?1, very weak scattering in the amide III region around 1250 cm^?1, and a medium-intensity line at 940 cm^?1. When the protein is pH-denatured, a strong amide III line appears at 1254 cm^?1 and the 940 cm^?1 line becomes weak. The intensities of the latter two lines are a sensitive measure of the alpha-helical and disordered chain content. These results are consistent with the helix-to-coil studies of the polypeptides. The Raman spectra of α-casein and prothrombin, proteins thought to have little or no ordered secondary structure, are investigated. The amide III regions of both spectra display strong lines at 1254 cm^?1 and only weak scattering is observed at 940 cm^?1, features characteristic of the denatured tropomyosin spectrum. The amide I mode of α-casein appears at 1668 cm^?1, in agreement with the previously reported spectra of disordered polypeptides, poly-L -glutamic acid and poly-L -lysine at pH 7.0 and mechanically deformed poly-L -alanine.     

The application of surface tension measurements to the study of conformational transitions in aqueous solutions of poly-L-lysine

The change in surface tension of solutions of poly-L -lysine in water has been studied as a function of temperature at various pH values. The changes at various temperatures have been correlated with changes in the circular dichroic spectra reflecting conformational change. In addition to the major transition at 50°C attributed to the conversion of the α-helical → β conformation, two other transitions have been observed at 30°C and 80°C. A minimum in the surface tension value was observed at pH 10, near the pK value for poly-L -lysine. It was concluded that at this pH the concentration of hydrophobic groups at the surface was a maximum.     

Single crystals of poly-L-lysine

Crystals of poly-L -lysine have been grown from aqueous solution in the presence of divalent anions. The most stable of these incorporate the HPO ion and are precipitated by the addition of sodium monohydrogen phosphate to solutions of poly-L -lysine HBr. Precipitation at or slightly above room temperature gives rise to single crystals of α-poly-L -lysine HPO₄ in the form of hexagonal lamellae about 150 Å thick. The axes of the helical polypeptide molecules are oriented normal to the planes of the lamellae, and since molecular length is about 1100 Å in the α-helical conformation, these helices must be folded. The a parameter of the hexagonal unit cell is 19.55 Å for crystals immersed in mother liquor, and the lysine side chains are almost fully extended. Precipitation brought about by heating the same solutions to about 75°C produces micro-crystals of β-poly-L -lysine HPO₄. A mode of packing of the anions in these crystals is proposed tentatively on the basis of an intersheet spacing determined from x-ray powder diffraction patterns. In general, α crystals are transformed to β structures on drying; conditions under which the transition can either be forestalled or reversed are discussed.     

Effect of temperature on the circular dichroism spectra of polypeptides in the extended state

The circular dichroism (CD) spectra of poly-L -proline and of poly-L -glutamic acid and poly-L -lysine in their charged states have been studied as a function of temperature. The variation of CD spectra with temperature is inconsistent with the assignment of the spectrum of such charged polypetides to an unordered chain conformation, but does support our earlier assignment to a locally ordered structure—what we have called the extended helix conformation. These results also strengthen our previous assignment of the CD spectrum of an unordered chain, and indicate that three conformational states (α-helix, extended helix, and unordered) should be incorporated in our thinking about conformational transitions in polypeptides.     

Raman scattering of some synthetic polypeptides: poly(gamma-benzyl L-glutamate), poly-L-leucine, poly-L-valine, and poly-L-serine

The Raman spectra of poly-γ-benzyl-L -glutamate, poly-L -leucine, poly-L -valine, and poly-L -serine are reported. For the α-helical polymers, the conformationally sensitive amide I, II, and III modes are observed in the Raman as, well as the infrared. For the β form, the Raman effect, supplies the infrared inactive inphase motion which is useful for the determination of a parallel or antiparallel chain alignment. Modes characteristic of the specific polypeptide are also observed which are insensitive to conformation.     

Volume and sound velocity changes accompanying the -helix to -form and coil to -helix transitions in aqueous solution

The volume increment per amino acid residue for the α-helix to β-form transition of uncharged poly-L -lysine in aqueous solution was 3.8 ml in water and 4.3 ml in 0.2M and 1M NaBr solutions at 26°C, respectively. The sound velocity of the polymer solution was greater with the β-helix than with the β-form, but the difference was less in dilute salt solutions and disappeared in 1 or 2M NaBr solution. Thus, the β poly-L -lysine solution was slightly more compressible than the α-polymer solution, but this difference was diminished with increasing salt concentration. Both the volume change and the change in adiabatic compressibility of the polymer solution suggest that hydrophobic interactions among the lysyl groups in the β-form reduce the amount of “icebergs” surrounding the polymer molecules as compared with the amount originally present with the α-helix. The coil-to-helix transition of poly-L -glutamic acid in aqueous solution was also accompanied by a decrease in sound velocity. This can be attributed to the reduction of the water of hydration which is less compressible than free water.     

Side-chain effect on conformation of ionizable polypeptides in aqueous solution

In order to study the effect of side-chain length on the conformation of polypeptides, conformational changes of various ionic polypeptides with various lengths of side chain, poly-N^ε-glutaryl-L -lysine (PGL), poly-N^δ-glutaryl-L -ornithine (PGO), poly-N^ε-succinyl-L -lysine (PSL), and poly-N^δ-succinyl-L -ornithine (PGO), were investigated by ORD, potentiometric titration, and dilatometric measurements in aqueous solution. The results of optical rotation and potentiometric titration measurements indicate strongly that the α-helix stability increases in the sequence PSO < PSL ～ PGO < PGL, which corresponds to increased side-chain length. The volume change associated with the helix–coil transition also increased in the above sequence. This series of polymers seems to be more hydrophobic compared with poly-L -glutamic acid or poly-L -lysine, as suggested from the values of enthalpy and entropy changes for coil–helix transitions.     

Optical properties of polypeptides in the β-conformation

The rotational strengths and oscillator strengths of the nπ* band and ππ* exciton bands have been calculated for antiparallel and parallel β-structures of varying length and width. The results are compared with experiment and with previous theoretical treatments of β-structures. The generally good agreement of calculations on the antiparallel β-structure with experimental results on poly-L -lysine and poly-L -serine indicates that these systems are indeed in the antiparallel conformation. It is found that the exciton component strongest in absorption shifts to longer wavelengths as the width of an antiparallel structure increases, and it is suggested that the position of the ππ* absorption band may be a useful criterion of sheet width. The results also reconcile the linear dichroism measurements of Rosenheck and Sommer on poly-L -lysine films with an anti-parallel structure. Calculations on parallel β-structures indicate that the CD spectra of this form will be rather similar to that of the antiparallel form. However, the major absorption band in the antiparallel form is associated with a small positive CD band, while in the parallel form it coincides with a large negative CD band. Finally, it is pointed out that the large positive CD bands predicted for single-stranded parallel and antiparallel β-structures at about 200 mμ render unlikely the suggestion that random-coil polypeptides contain a substantial fraction of extended chain.     

Conformational studies on polypeptides. The effect of sodium perchlorate on the conformation of poly-L-lysine and of random copolymers of L-lysine and L-phenylalanine in aqueous solution

Circular Dichroism measurements have been carried out on poly-L -lysine (PLL) and on random copolymers of lysine and phenylalanine at various p^H values and in the presence of different amounts of NaClO₄. The results indicate that either the homopolymer or the copolymers at p^H conditions at which the side-chain amino groups are fully protonated, assume the right-handed α-helical conformation in the presence of NaClO₄. The results are interpreted in terms of specific binding of ClO₄^? ions on charged side-chain amino groups.     

Low-frequency vibrational spectra of some homopolypeptides in the solid state

Low-frequency Raman and far-infrared spectra of polyglycine, poly-L -alanine, and poly-L -valine have been measured. The Raman spectra exhibit an intense band near 100 cm^?1 for these homopolypeptides. Lattice calculations of the polyglycines are used to assign the intense Raman band to a rotary lattice mode. For homopolypeptides in the β conformation, an infrared band is observed whose frequency varies inversely with the square root of the mass of the peptide repeating unit. This infrared band is assigned to the hydrogen bond stretching lattice vibration.     

Hydration mobility in peptide structures

The structural behavior of hydrochlorides of poly-L -lysine and tetraglycine depends on water vapor pressure. At low relative humidities, structural rearrangements are slow. Water molecules catalyze these structural rearrangements; thus, in tetraglycine 1 H²O molecule per 10 peptide residues. Some general aspects of the mechanism of the hydration mobility in peptide structures are discussed.