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.     

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.     

Morphology and structure of γ-benzyl-l-glutamate copolymerized with l-phenylalanine, l-valine and l-alanine

Observation of random copolypeptides of γ-benzyl-l-glutamate with l-phenylalanine, l-valine and l-alanine was carried out in an electron microscope with samples cast from dilute solution. The relationship between the morphology and the molecular conformation in solution was studied with mixed solvents composed of chloroform and trifluoroacetic acid; these show a preference for α-helix and random coil, respectively. From the solutions in which molecules take α-helical conformation, fibrous films of nematic structure were formed. From random coil solutions discrete precipitates with folded molecules such as lamellar single crystals, piles of lamellae and structureless particles were formed. A copolypeptide containing l-valine in sufficiently large quantity to form β-structure also showed a variation in morphology with solvent, from films to discrete precipitates. It is suggested that the change in stiffness of the molecules contributes to the morphological variation.     

Conformational studies of alternating copoly(Leu-Lys) and copoly(Leu-Orn) in alcohol-water solvent mixtures

Conformational transitions of alternating copoly(l-leucyl-l-lysine) and copoly(l-leucyl-l-ornithine) in organic solvents and in alcohol-water mixtures were determined by c.d. measurements and the results compared with those from random copoly(Leu^48.3, Lys^51.7). As reported previously^16,17, in salt-free water these alternating copolymers undergo a conformational transition from a disordered to β-structure when the pH is raised or when various salts are added, whereas random copolymers adopt an α-helix conformation under similar conditions. However, both alternating copolymers reveal a tendency to form α-helix in 2,2,2-trifluoroethanol and in alcohol-water mixtures at neutral pH, as does the random copolymer. The alcohol concentration at which the α-helix can be induced is dependent on the kind of alcohol, the α-helix promoting power follows the the series: 2,2,2-trifluoroethanol > isopropanol > ethanol > methanol. In addition, these alternating copolymers in methanol-water mixtures below 50% (by volume) methanol form the β-structure when the pH is raised. On the other hand, above 60% methanol the fraction of α-helix already formed at neutral pH is enhanced at higher pH-values.     

Conformational studies of poly(L-tyrosine). A helix–coil transition in dimethyl sulfoxide–dichloroacetic acid mixtures

Poly(L -tyrosine) is a random coil in dimethyl sulfoxide. Upon addition of dichloroacetic acid, poly(L -tyrosine) undergoes a conformational transition centered at about 10% dichloroacetic acid. The transition is nearly complete at 20% dichloroacetic acid. Further addition of dichloroacetic acid leads to precipitation of poly(L -tyrosine). We have characterized this transition by optical rotation, viscosity, circular dichroism, and infrared. The optical rotation at 350 nm and the intrinsic viscosity increase sharply to values that are consistent with a transition to the α-helix conformation. The circular dichroism of poly(L -tyrosine) in dimethyl sulfoxide and in dimethyl sulfoxide/dichloroacetic acid (80:20 v/v) agrees with previous reports for random-coil and α-helix conformations, respectively. The infrared spectrum of poly(L -tyrosine) in dimethyl sulfoxide/dichloroacetic acid (80:20 v/v) shows no evidence of β-structure. We conclude that the transition on going from dimethyl sulfoxide to 20% dichloroacetic acid in dimethyl sulfoxide is a coil → α-helix transition. The amide-I band of poly(L -tyrosine) in dimethyl sulfoxide/dichloroacetic acid (80:20) is found to be at 1662 cm^?1. It has been suggested that this high frequency may be indicative of a left-handed α-helix. However, this high amide-I frequency is consistent with conformational energy calculations of Scheraga and co-workers. The mechanism of the dichloroacetic acid-induced transition to an α-helix is discussed. Dichloroacetic acid and dimethyl sulfoxide interact strongly and the transition presumably involves a marked decrease in the ability of dimethyl sulfoxide to solvate the peptide backbone and aromatic side chains upon complex formation with dichloroacetic acid.     

The aggregation of basic polypeptide residues bound to heparin

The molecular basis for heparin interactions with proteins has been explored with l-lysine copolymer: heparin complexes, measuring the conformational change and charge neutralization which accompany the complexation, using optical methods. Previous studies had shown that the basic homopolypeptides (poly-l-lysine, poly-l-arginine) assume α-helical conformation upon interaction with numerous glycosaminoglycans (including heparin). Thus, the unique specificity for heparin in the anticoagulation system (which involves two or more lysine residues on the antithrombin molecule) is not paralleled by the findings with the basic homopolymers.Results with mixed polypeptides, poly(lysine: tyrosine, 1:1) and poly(lysine: phenylalanine, 1.4:1), show that these protein models assume different conformational forms upon complexation with heparin, the former shows a poly-l-lysine-like β-structure circular dichroism spectrum and the latter an α-helical structure. The change in circular dichroism spectra increases with the addition of heparin until the ratio of positive to negative charge is about one. Dye-binding studies of the two copolymer systems reveal that the charged groups of reactants are largely blocked in the polypeptide complexes at a calculated charge ratio equal to one. The data indicate that heparin interaction with the cationic polypeptides causes them to assume either the α-helical or β-structure depending upon the nature of the neighboring uncharged amino acid and its proclivity for α-helix or β-structure.     

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.     

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     

Structural changes of IgG induced by heat treatment and by adsorption onto a hydrophobic Teflon surface studied by circular dichroism spectroscopy

Thermal denaturation of mouse monoclonal immunoglobulin G (isotype 1), as well as structural rearrangements resulting from adsorption on a hydrophobic Teflon surface, are studied by circular dichroism spectroscopy. Both heat-induced and adsorption-induced denaturation do not lead to complete unfolding into an extended polypeptide chain, but leave a significant part of the IgG molecule in a globular or corpuscular form. Heating dissolved IgG causes a decrease of the fractions of β-sheet and β-turn conformations, whereas those of random coil and, to a lesser extent, α-helix increase. Adsorption enhances the formation of α-helices and random coils, but the β-sheet content is strongly reduced. Heating adsorbed IgG results in a gradual break-down of the α-helix and β-turn contents, and a concomitant formation of β-sheet structures. Thus, the structural changes in IgG caused by heating and by adsorption, respectively, are very different. However, after heating, the structure of adsorbed IgG approaches the structure of thermally denatured IgG in solution.     

Kinetics of coil to α-helix to β-structure transitions of poly(L-ornithine) in low concentrations of sodium dodecyl sulfate

Conformational changes of poly(L-ornithine) [(Orn)_n] were studied in a sodium dodecyl sulfate (NaDodSO₄) solution by CD. (Orn)_n adopted an unstable and a stable helical structure below and above the NaDodSO₄ concentration range where β-structure was favored, respectively. CD stopped-flow was used to monitor the transitions from coil to the unstable helix, from the helix to β-structure, and from coil to β-structure. Only the rate of the helix to β-structure transition was accelerated by an increase in NaDodSO₄ concentration, whereas the rates of the others were independent of NaDodSO₄ concentration. The fractions of coil, α-helix, and β-structure in each conformation of (Orn)_n caused by NaDodSO₄ were computed by simulating a mixed spectrum of typical CD spectra for these structures to the experimentally obtained spectrum. The contents of the unstable and stable helical structures were less than 50 and 73%, respectively.     

Homooligopeptides composed of hydrophobic amino acid residues interact in a specific manner by taking α-helix or β-structure toward lipid bilayers

In order to investigate the role of each amino acid residue in determining the secondary structure of the transmembrane segment of membrane proteins in a lipid bilayer, we made a conformational analysis by CD for lipid-soluble homooligopeptides, benzyloxycarbonyl-(Z-) Aaa_n-OEt (n = 5-7), composed of Ala, Leu, Val, and Phe, in three media of trifluoroethanol, sodium dodecyl sulfaie micelle, and phospholipid liposomes. The lipid-peptide interaction was also studied through the observation of bilayer phase transition by differential scanning cahrimetry (DSC). The CD studies showed that peptides except for Phe oligomers are present as a mainly random structure in trifluoroethanol, as a mixture of α-helix, β-sheet, β-turn, and /or random in micelles above the critical micellization concentration and preferably as an extended structure of α-helical or β-structure in dipalmitoyl-D,L -α-phosphatidylcholine (DPPC) liposomes of gel state. That the β-structure content of Val oligomers in lipid bilayers is much higher than that in micelles and the oligopeptides of Leu (n = 7) and Ala (n = 6) can take an α-helical structure with one to two turns in lipid bilayers despite their short chain lengths indicates that lipid bilayers can stabilize the extended structure of both α-helical and β-structures of the peptides. The DSC study for bilayer phase transition of DPPC / peptide mixtures showed that the Leu oligomer virtually affects neither the temperature nor the enthalpy of the transition, while Val and Ala oligomers slightly reduce the transition enthalpy without altering the transition temperature. In contrast, the Phe oligomer affects the phase transition in much more complicated manner. The decreasing tendency of the transition enthalpy was more pronounced for the Ala oligomer as compared with the Leu and Val oligomers, which means that the isopropyl group of the side chain has a less perturbing effect on the lipid acyl chain than the methyl group of Ala. © 1995 John Wiley & Sons, Inc.     

A stone's throw and its launch window: timing precision and its implications for language and hominid brains

The Chou-Fasman conformational parameters, P, for amino acid residues in proteins are shown to be a linear function of intermolecular force and steric parameters. For α- helix, coil and turn parameters, steric effects are predominant; whereas for β-sheet parameters, intramolecular forces are predominant. Turn and coil parameters show little or no difference in their dependence which is different from that of α-helix and in some ways almost reciprocal. Factors which increase the probability of finding an amino acid residue in an α-helix usually decrease the probability of finding it in coil or turn. Values of P were calculated for several of the less common amino acids.     

Binary patterning of polar and nonpolar amino acids in the sequences and structures of native proteins

Protein sequences can be represented as binary patterns of polar (○) and nonpolar (?) amino acids. These binary sequence patterns are categorized into two classes: Class A patterns match the structural repeat of an idealized amphiphilic α-helix (3.6 residues per turn), and class B patterns match the structural repeat of an idealized amphiphilic β-strand (2 residues per turn). The difference between these two classes of sequence patterns has led to a strategy for de novo protein design based on binary patterning of polar and nonpolar amino acids. Here we ask whether similar binary patterning is incorporated in the sequences and structures of natural proteins. Analysis of the Protein Data Bank demonstrates the following. (1) Class A sequence patterns occur considerably more frequently in the sequences of natural proteins than would be expected at random, but class B patterns occur less often than expected. (2) Each pattern is found predominantly in the secondary structure expected from the binary strategy for protein design. Thus, class A patterns are found more frequently in α-helices than in β-strands, and class B patterns are found more frequently in β-strands than in α-helices. (3) Among the α-helices of natural proteins, the most commonly used binary patterns are indeed the class A patterns. (4) Among all β-strands in the database, the most commonly used binary patterns are not the expected class B patterns. (5) However, for solvent-exposed β-strands, the correlation is striking: All β-strands in the database that contain the class B patterns are exposed to solvent. (6) The bias of class A patterns for α-structure over β-structure and the bias of class B patterns for β-structure over α-structure are significant, not merely when compared to other binary patterns of polar (○) and nonpolar (?) amino acids, but also when compared to the full range of sequences in the database. The implications for the design of novel proteins are discussed.     

Existence of γ-helix in natural proteins

Serum albumin is known to act as a carrier for a variety of molecules and metal ions. This property of the protein could be due to the presence of different types of secondary structures in its molecules. The most commonly known are the α-helix, β-sheets, β-turns and random coil forms. A rigorous analysis of human serum albumin has been carried out by using four different approaches. Comparative studies have revealed that the segment Asp 107 to Val 122 of this protein assumes a γ-helical structure. Under favourable circumstances, two prolines at the i and (i + 5)th positions can initiate a γ-helix. Further requirements for the formation and stability of γ-helix are discussed.     

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.     

Adsorption of polypeptides on a solid surface. IV. Adsorption and β-structure–coil transition

A theory of adsorption of a polypetide chain capable of undergoing the coil–β-structure transition on a solid planar surface has been developed. The mutual influence of two order–disorder phase transitions, a conformational and an adsorption transition, was investigated. Various types of adsorption transitions are possible, depending on the initial conformational state (partly or completely β-structured) and the selectivity of adsorption: (a) the second-order phase transition, in which the chain is partly structured, both in adsorbed and desorbed states; and (b) the first-order phase transition, in which the chain exhibits a regular β-structure, at least on one side of the adsorption transition boundary. The chain bonding to the surface alters the degree of β-structure, both in the case of selective and nonselective adsorption (similar to the adsorption of the chains with other types of secondary structure). We show that the slope of the adsorption curves for partly β-structural chains increases as a result of an increase in the degree of β-structuring, and this effect is even stronger than the analogous effect of β-structuring.     

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.     

Blood glycoprotein from antarctic fish possible conformational origin of antifreeze activity

High resolution ¹H NMR and circular dichroism (CD) measurements have been performed on aqueous solutions of antarctic fish antifreeze glycoprotein. The carbohydrate contribution to the observed CD spectrum has been estimated from closely analogous model compounds. The residual peptide contribution cannot be interpreted of the known spectral behaviour of α-helix, β-sheet and random coil. Instead it resembles the CD spectrum of β-structure in position, magnitude and spectral form, but is of opposite sign, indicating a specific but unusual peptide conformation, which we suggest may be stabilized by non-bonded interactions between the peptide backbone and the carbohydrate sidechains. Previous evidence which supports this interpretation is reviewed. NMR and CD measurements between ?2 and +30°C are consistent with conformational stability throughout the biologically relevant temperature range. The mechanism of the antifreeze activity is discussed in terms of the spatial and orientational correlations of sugar hydroxy groups and water in the liquid and solid states. The implication of an ordered peptide structure is explained by the comparison of the antifreeze glycoprotein with synthetic water-soluble polymers which also exhibit limited antifreeze properties.     

Conformation of poly(l-glutamic acid) at the air/water interface

The conformation of the monolayer of poly(l-glutamic acid) on subsolutions of different pH values was studied by the film-balance technique, obtaining surface pressure measurements, together with polarized infrared spectroscopy and Raman spectroscopy. The monolayers of poly(l-glutamic acid) gave different surface pressure-area curves on subsolutions of various pH values. It was found that the conformation of poly(l-glutamic acid) monolayer spread at the air/water interface differs from that in solution. It can be presumed that poly(l-glutamic acid) in a monolayer is in the form of an α-helix at pH 2.0, in the β-form at pH 3.5 and in an ‘intramolecular’ heterogeneous conformation (consisting of a random coil and an α-helix) at pH 4.0.     

Fourier transform IR attenuated total reflectance study on the secondary structure of poly(γ -methyl L-glutamate) surfaces treated with formic acid

Fourier transform ir attenuated total reflectance (FTIR ATR) spectra have been obtained to investigate the secondary structure of poly(γ -methyl L -glutamate) (PMLG) surfaces untreated and treated with formic acid in a quantitative manner. Curve analysis including Fourier self-deconvolution and the band fitting was applied to the ir spectra in the amide II region, revealing that the amide II band of those surfaces consists of five components. The essentially α-helical form in the PMLG surface layer transformed readily into the β-structure by the formic acid treatment, and the β-structure content increased with increasing time of the treatment. The content of random coil structure of treated PMLG was generally very little and/or negligible. The depth profile obtained by considering the depth of ir beam penetration indicated that the β-structure content also increased with approaching the outermost surface.