Conformational properties of L-leucine,L-isoleucine,and L-norleucine side chains in L-lysine copolymers

The structure inducing properties of L -leucine, L -isoleucine, and L -norleucine residues incorporated into poly(L -lysine) were investigated by the observation of the circular dichroism of the respective random copolypeptides. The comparison involves the coil-helix transition in water/methanol mixtures, the formation of ordered structures at higher pH, and the kinetics of the α-helix to β-conformation transition of the leucine and norleucine copolymers induced by temperature changes at pH 10.5. The results confirm the known properties of the leucine residue, strongly supporting the α-helix conformation. They also support the idea that the isoleucine residue is one of the most powerful candidates for β-structure formation, and they show that the unbranched norleucine residue has intermediate properties. The results are discussed on the basis of steric and hydrophobic properties of the three side chains.     

Raman spectra of copoly(D,L-alanines)

Raman spectra in the region 1000–150 cm^?1 were measured for copoly(D ,L -alanines) with the D -residue contents, 3, 7, 10, and 20%, and compared with the spectrum of the α-helical poly-L -alanine. The 532- and 378-cm^?1 peaks were assigned to the L -residues with a right-handed α-helix-like local conformation or to the D -residues with a left-handed α-helix-like local conformation. From the intensity of the latter peak the contents of these local conformations were estimated as a function of the D -residue contents for the copolymers. The 264-cm^?1 peak, which has been assigned to the breathing vibration of the α-helical poly-L -alanine, shows a marked decrease in its intensity upon the introduction of the D residues. This result suggests that the overall deformation vibration of the α-helix arises from rather long sequences of the L - and D -alanine residues with the α-helical conformation and that the intensity of this vibration depends on the content of these sequences in the copolymers.     

Poly(L-lysyl-L-alanyl-alpha-L-glutamic acid). II. Conformational studies.

The solution characterization of poly(Lys-Ala-Glu) is described. This polytripeptide is zwitterionic at neutral pH and is shown to take on a conformation which is dictated by the state of ionization, molecular weight, temperature, and solvent. The polypeptide is almost entirely α-helical at low pH and temperature for polymers of greater than 25,000 molecular weight. Melting profiles for these conditions show t_m ～ 20°C. Analysis of circular dichroism curves shows the α-helical content to vary in a linear manner with molecular weight in the range 3000–30,000. At neutral pH the charged polypeptide is essentially random, but substantial α-helix could be induced by addition of methanol or trifluoroethanol. At temperatures where the sequential polypeptide is a random coil, addition of trifluoroethanol produces a polymer which is mostly α-helical but also contains an appreciable ammount of β-structure. The infrared spectrum of a low-molecular-weight fraction assumed to be cyclo(Lys-Ala-Glu)₂ was tentatively assigned a β-pleated sheet structure. A comparison of this polytripeptide in various ionization states with other polytripeptides containing L -alanine and L -glutamate or L -lysine shows the α-helix directing properties for the (uncharged) residues to lie in the order Ala > Glu > Lys.     

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.     

Time-dependent Structure and Activity Changes of α-Chymotrypsin in Water/Alcohol Mixed Solvents

Secondary structure of α-chymotrypsin in water/ethanol was investigated by circular dichroic (CD) spectroscopy. The changes in catalytic activity were discussed in terms of structural changes of the enzyme. α-Chymotrypsin formed β-sheet structure in water/ethanol (50/50 by volume), but it was substantially less active as compared to that in water. At water/ethanol 10/90, α-chymotrypsin took on a native-like structure, which gradually changed to β conformation with concomitant loss of activity. Change of solvent composition from water/ethanol 50/50 to 90/10 or 10/90 by dilution with water or ethanol, respectively, led to partial recovery of native or native-like structure and activity. In water/methanol, α-chymotrypsin tended to form stable β-sheet structure at water/methanol ratios lower than 50/50, but the catalytic activity decreased with time. Change to α-helix structure with substantial loss in catalytic activity was observed when α-chymotrypsin was dissolved in water/2,2,2-trifluoroethanol with water contents lower than 50%. In water/2,2,2-trifluoroethanol 90/10, α-chymotrypsin initially had the CD spectrum of native structure, but it changed with time to that characteristic of β-sheet structure.     

Singular value decomposition analysis of the secondary structure features contributing to the circular dichroism spectra of model proteins

Amyloid fibril formation occurs in restricted environment, such as the interface between intercellular fluids and bio-membranes. Conformational interconversion from α-helix to β-structure does not progress in fluids; however, it can occur after sedimentary aggregation during amyloid fibril formation induced by heat treatment of hen egg white lysozyme (HEWL). Secondary structures of various proteins and denatured proteins titrated with 2,2,2-trifluoroethanol (TFE) were examined using their CD spectra. Gaussian peak/trough and singular value decompositions (SVD) showed that the spectral pattern of the α-helix comprised a sharp trough at wavelength 207 nm and a broad trough at 220 nm. Conversely, we distinguished two patterns for β-sheet—a spread barrel type, corresponding to ConA, and a tightly weaved type, corresponding to the soybean trypsin inhibitor. Herein, we confirmed that the spectral/conformational interconversion of the heat-treated HEWL was not observed in the dissolved fluid.     

Reversible effects of medium dielectric constant on structural transformation of β-lactoglobulin and its retinol binding

The secondary structure transformation of β-lactoglobulin from a predominantly β-structure into a predominantly α-helical one, under the influence of solvent polarity changes is reversible. Independent of the alcohol used — methanol, ethanol, or 2-propanol — the midpoints of the observed structural transformation occur around dielectric constant ε ≈ 60. The structural change destroying the hydrophobic core formed by the β-barrel structure leads, at room temperature, to the dissociation of the retinol/β-lactoglobulin complex in the neighborhood of dielectric constant ε ≈ 50. However, when the dielectric constant of the medium is raised back to ε ≈ 70 by the decrease of the temperature, both the refolding of BLG into a β-structure and the reassociation of the retinol/β-lactoglobulin complex are observed. The esterification of β-lactoglobulin carboxyl groups has two effects: on the one hand it accelerates the β-strand → α-helix transition induced by alcohols. On the other hand, the esterification of β-lactoglobulin strengthens its interaction with retinol as it may be deduced from the smaller apparent dissociation constant of retinol/methylated β-lactoglobulin complex. The binding of retinol to modified or unmodified β-lactoglobulin has no influence (stabilizing or destabilizing) on the folding changes induced by alcohol. © 1993 John Wiley & Sons, Inc.     

Nature of amino acid side chain and alpha-helix stability.

In order to investigate the ability of neutral amino acids to support the α-helix conformation, the coil–helix transition of poly(L -lysine) and of lysine copolymers with these amino acids was studied in water/methanol using circular dichroism. The transtions were recorded at constant pH adding buffer to the methanol/water mixtures. With poly(L -lysine), experiments were performed at several constant pH's; the transition midpoint on the water (methanol) concentration scale was found to depend strongly upon pH; the helix stability region is shifted towards higher water concentrations, when the pH is increased. Copolymers of lysine and several neutral amino acids revealed the same effect in that increasing amounts of, for example, norleucine also shifted the transition midpoint to higher water concentrations. A series of copolymers containing L -lysine as the host and different hydrophobic amino acids were synthesized and the helix–coil transition in water/methanol was observed at constant pH. Different copolymers of equal composition showed significant differences with respect to the nature of the amino acid incorporated into polylysine. From these studies an α-helix-philic scale (in decreasing order): Leu, Nle, Ile, Ala, Phe, Val, Gly is deduced and discussed; the results obtained were compared with those of different procedures.     

Conformational aspects of gastrin-related peptides: A circular dichroism study

The conformational properties of a series of gastrin-related peptides in aqueous solution and in 2,2,2-trifluoroethanol (TFE) have been investigated by CD measurements. In aqueous solution the peptides Leu³²-HG-34 (human big gastrin), Nle¹⁵-HG-17 (human little gastrin), and Nle¹¹-HG-13 assume a random-coil structure in the pH range 3–7. In TFE the three hormones fold into partially ordered structures, consisting of mixtures of α-helix, β-form and random coil. Comparison with the CD properties of the shorter gastrin peptides HG-4 (tetragastrin), N^α-Boc-HG-5 (pentagastrin), and HG-7 (heptagastrin) indicates that the biologically important C-terminal sequence Trp-Met-Asp-Phe-NH₂ in TFE does not maintain the same geometry upon elongation of the chain at the N-terminus from 4 to 34 residues. Thus, the various conformations in solution of the gastrin peptides examined do not provide a structural explanation for their very similar biological activity. Therefore, we hypothesize that the C-terminal tetrapeptide amide folds into an “active” structure only upon interaction with the receptor.     

Hydrodynamic behavior and molecular conformation of poly(L-lysine HBr) in carbonate buffer solution

In carbonate buffer at pH 10.5, a transparent solution of poly(L -lysine HBr) was obtained up to fairly high concentration of 3 g/dl at room temperature. The hydrodynamic behavior of the solution has been studied by sedimentation analyses and viscosity measurements. A dimer form for high concentrations and a monomer form for low concentrations were inferred. The dimer and monomer forms were assigned to a β-structure and α-helix, respectively, based on the CD and optical rotary dispersion spectra. Using CD spectroscopy, a reversible transition between α-helix and β-structure was observed as a function of either poly(L -lysine HBr) concentration or temperature. An aggregated form which was assigned to the antiparallel pleated sheet appeared at 50°C on the basis of its ir spectrum.     

Photoresponsive polymers: Azobenzene-containing poly(L-lysine)

Poly(L -lysine) was reacted with various azo-reagents, including p-phenylazobenzoic acid, p-phenylazobenzoyl chloride, and p-phenylazobenzoic N-hydroxy-succinimide ester, to give polypeptides containing 5–44 mol % azobenzene units in the side chains. The conformation of the azo-modified polypeptides was investigated in connection with their photochromic behavior caused by the trans ? cis photoisomerization of the azo groups present in the side chains. In methanol/water solvent mixture, the 20% azo-poly(L -lysine) adopts the α-helix conformation. The helix stability was found to be higher when the azo side chains are in cis than when they are in trans configuration. So irradiation at 340 nm (trans-to-cis isomerization), and alternately at 450 nm (cis-to-trans isomerization), produced reversible variations of the α-helix content. In hexafluoro-2-propanol/water/sodium dodecyl sulfate mixture, the 43% azo-poly(L -lysine) adopts a β-structure, as indicated by CD spectra. Irradiation at 340 nm caused the disruption of the β-structure and promoted the α-helix conformation. The effect was reversed upon irradiation at 450 nm. The photoinduced β ? helix change was explained on the basis of the different geometry and hydrophobic character of the trans and the cis azobenzene units.     

Potentiometric titration of poly-L-lysine: the coil-to-beta transition

We have performed potentiometric titrations of poly-L -lysine. From these data we have calculated the free energy and enthalpy changes for the folding of the random coil to the α-helix in 10% ethanol (?120 and ?120 cal/mole) and from the random coil to the β-structure in water (?140 and 870 cal/mole) and in 10% ethanol (?180 and 980 cal mole). Comparison of these values with each other and with values for the coil → α- helix transition in water (?78 and ?880 cal/mole) led to the following conclusions. The stabilization by ethanol of ethanol of the α-helix with respect to the coil is that predicted from the known free energy of transfer of the peptide group from water to 10% ethanol. Similar data to explain the enthalpy difference are not available. The thermodynamic functions for the transition from α-helix to β-structure, obtained by subtracting those for the coil → α-helix and coil → β-structure transitions, are explained from a consideration of the structural differences: non bonded interactions of the polypeptide backbone are less favorable in the β-structure than in the α-helix, causing an increase in the energy, while hydrophobic contacts between side chains raise the entropy of the β-structure as compared with the α-helix, so that the free energy difference between the two structures is small, but enthalpy and entropy differences are large. The observation of only small differences in the free energy and enthalpy changes for the transition from coil β-structure upon going from water to 10% ethanol is expected by considering both the free energy of transfer of the peptide group (as for the α-helix) and the free energy and enthalpy of transfer of the apolar part of the side chain involved in hydrophobic bond formation.     

Conformation of sequential and random lysine–phenylalanine copolypeptides

The sequential copolypeptides (Lys-Phe-Lys)_n and (Lys₂-Phe-Lys)_n and a series of related random copolypeptides were investigated with respect of their ability to adopt the α-helix or β-conformation. Conformational transitions were induced by increasing the pH or by addition of NaClO₄ or methanol and were observed by recording the CD spectra. In contrast to the respective alternating copolypeptide (Phe-Lys)_n with its strong tendency for the β-structure reported previously, (Lys-Phe-Lys)_n can adopt either secondary structure, whereas (Lys₂-Phe-Lys)_n strongly favors the α-helix. Together with the random copolypeptides, whose composition varied from 20 to 50 mol % phenylalanine and whose average molecular weights ranged from 10,000 to 90,000, the influence of the phenylalanine content and of the chain length on conformational stability and the rotatory strength of the respective secondary structures were elaborated.     

Calculation of the molecular parameters of the alpha-helix-coil and beta-structure-coil transitions.

Monte-Carlo calculations of geometric and thermodynamic characteristics of the α-helix and the β-structure of polypeptides have been carried out. To describe a hydrogen bond both the Lippincott–Schroeder and Morse potentials were used. The internal rotation angles φ and ψ in the α-helix have been shown to fluctuate in the range of ±7°. The distribution functions on angles φ and ψ and on hydrogen bond lengths and angles in the α-helix have been computed and compared with those in myoglobin and lysozyme. Thermodynamic characteristics of the α-helix calculated in different approximations with the two forms of the hydrogen bond potentials have also been compared. The data obtained are close to the experimental values for polypeptides in neutral solution. Some geometric and thermodynamic characteristics of the regular parallel and antiparallel and irregular antiparallel β-structure have been found. In the β-structure the internal rotation angles vary within the interval ±15–20°. An increase in the cross and longitudinal dimensions of the β-structure only slightly influence both the geometric and thermodynamic characteristics.     

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     

Effect of alcohols on the structure of caseins: circular dichroism studies of κ-casein A

The alcohols methanol, ethanol, propan-1-ol and 2,2,2-trifluoroethanol were added to aqueous solution of κ-casein A. Far u.v. circular dichroism spectroscopy revealed a greater proportion of -helix structure in the presence of alcohol; the effectiveness of the alcohol in causing this structural change increased in the order methanol to 2,2,2-trifluoroethanol. No alterations in secondary or tertiary structure were found in κ-casein A on reduction and S-carboxymethylation using near and far u.v.c.d. spectroscopy. The isolated macropeptide section of κ-casein was less sensitive to the addition of alcohols; it appeared that the linkage of macropeptide to the para-κ-casein section affected the case with which solvent induced conformational changes occurred. The conformational changes found in the presence of alcohols could not be correlated simply with the changes in the dielectric constant or with the alcohol-induced collapse of the hairy macropeptide layer of casein micelles or ther precipitation.     

Intramolecular conformational transitions "random coil-helix-folded structure" in polypeptides.

A general theory has been developed for conformational intramolecular transitions in a single macromolecule with a high degree of polymerization (an infinite length model) capable of forming two types of ordered structures: the α-helix and the folded β-structure, as well as acquiring the random coil conformation. The phase diagram analysis of this system has shown that the regular β-structure state is separated from all other states of the chain by the phase boundary line. Any intersection of the phase boundary is a phase transition which can be either of the first order or second order, depending on values of the energy parameters of the system. Mechanisms of intramolecular rearrangements: β-structure–random coil and α-helix–β-structure have been discussed. It has been shown that there exist two different mechanisms for each of these rearrangements, and the regions of parameter variation corresponding to each mechanism have been specified.     

The infuence of interchain compositional heterogeneity on the conformation in random copolymers of γ-benzyl-L-glutamate and L-valine

Variation in the solvent used for the copolymerization of γ-benzyl-L -glutamate and L -valine N-carboxyanhydrides provides copolymers which have variable interchain compositions, and this variation in interchain compositional heterogeneity is reflected in the solid-state conformations of the respective copolymers. Poly[Glu(OBzl)²⁹Val⁷¹] prepared in dioxane exhibits a β-structure, whereas a copolymer of the same average composition prepared in benzene/methylene chloride shows predominantly an β-helix conformation with a small amount of β-structure. The use of the monomer reactivity ratio permits the calculation of the average and incremental copolymer compositions at any conversion; thus, correlations between conformation and interchain compositional heterogeneity can be made. In general, copolymers prepared in dioxane show a greater distribution of chain composition and therefore permit a wider variety of conformation than copolymers prepared in benzene/methylene chloride under identical conditions.     

Extent and stability of the alpha-helix in gamma-benzyl L-glutamate-glycine copolymers

Block and random copolymers of γ-benzyl L -glutamate and glycine were studied by optical rotatory dispersion methods in order to ascertain the extent and stability of the α-helix formed by these polymers in appropriate solvent mixtures. Results indicate that when in solution such polymers do contain sections of α-helix. However, it appears that glycine does not readily fit into the α-helix which is largely due to the γ-benzyl L -glutamate segments. Further, there are indications that the helical sections in the random copolymers are due to the nonrandomness of the polymerization.     

Conformational changes of α-lactalbumin and its fragment,Phe31-Ile59, induced by sodium dodecyl sulfate

Conformational changes of bovine α-lactalbumin in sodium dodecyl sulfate (SDS) solution were studied with the circular dichroism (CD) method using a dilute phosphate buffer ofpH 7.0 and ionic strength 0.014. The proportions of α-helix and β-structure in α-lactalbumin were 34% and 12%, respectively, in the absence of SDS. In the SDS solution, the helicity increased to 44%, while the β-structure disappeared. In order to verify the structural change from β-structure to α-helix, the moiety, assuming the β-structure in the α-lactalbumin, was isolated by a chymotryptic digestion. The structure of this α-lactalbumin fragment, Phe³¹-Ile⁵⁹, was almost disordered. However, the fragment adopted a considerable amount of α-helical structure in the SDS solution. On the other hand, the tertiary structure of α-lactalbumin, detected by changes of CD in the near-ultraviolet region, began to be disrupted before the secondary structural change in the surfactant solution. Dodecyl sulfate ions of 80 mol were cooperatively bound to α-lactalbumin. Although the removal of the bound dodecyl sulfate ions was tried by the dialysis against the phosphate buffer for 5 days, 4 mol dodecyl sulfates remained per mole of the protein. The remaining amount agreed with the number of stoichiometric binding site, determined by the Scatchard plot, indicating that the stoichiometric binding was so tight.