Interaction of amphipathic polypeptides with phospholipids: characterization of conformations and the CD of oriented beta-sheets

The effects of interaction with phospholipids on the conformation of the alternating copolymer, poly(Leu-Lys), and the random copolymer poly(Leu⁵⁰, Lys⁵⁰) have been investigated by CD and ir spectroscopy. Poly(Leu-Lys) undergoes a partial unordered → β-sheet transition in solution in the presence of lysolecithin. On addition of lysolecithin plus cholate, an unordered → α -helix transition is observed. In films deposited from these solutions, poly(Leu-Lys) adopts the anti-parallel β-sheet conformation, as in aqueous solutions at moderate ionic strength. Polarized ir spectra showed that the plane of the β-sheet in such films deviates from the plane of the film by no more than 14°. The random copolymer, poly(Leu⁵⁰, Lys⁵⁰), is α-helical in the presence of lysolecithin and lysolecithin plus cholate, regardless of whether the sample is a solution or a film. CD measurements on the poly(Leu-Lys) films provide information about the component of the CD tensor for light propagating normal to the plane of the β-sheet. These measurements show (1) a negative n → π* CD band (214 nm maximum) with higher intensity than the average CD for isotropic solution; and (2) a positive band in the π → π* region (195 nm maximum), which is weaker than that in the isotropic spectrum.     

Secondary structure of proteins associated in thin films

The solid state secondary structure of myoglobin, RNase A, concanavalin A (Con A), poly(L -lysine), and two linear heterooligomeric peptides were examined by both far-uv CD spectroscopy¹ and by ir spectroscopy. The proteins associated from water solution on glass and mica surfaces into noncrystalline, amorphous films, as judged by transmission electron microscopy of carbon-platinum replicas of surface and cross-fractured layer. The association into the solid state induced insignificant changes in the amide CD spectra of all α-helical myoglobin, decreased the molar ellipticity of the α/β RNase A, and increased the molar ellipticity of all-β Con A with no change in the positions of the bands' maxima. High-temperature exposure of the films induced permanent changes in the conformation of all proteins, resulting in less α-helix and more β-sheet structure. The results suggest that the protein α-helices are less stable in films and that the secondary structure may rearrange into β-sheets at high temperature. Two heterooligomeric peptides and poly (L -lysine), all in solution at neutral pH with “random coil” conformation, formed films with variable degrees of their secondary structure in β-sheets or β-turns. The result corresponded to the protein-derived Chou-Fasman amino acid propensities, and depended on both temperature and solvent used. The ir and CD spectra correlations of the peptides in the solid state indicate that the CD spectrum of a “random” structure in films differs from random coil in solution. Formic acid treatment transformed the secondary structure of the protein and peptide films into a stable α-helix or β-sheet conformations. The results indicate that the proteins aggregate into a noncrystalline, glass-like state with preserved secondary structure. The solid state secondary structure may undergo further irreversible transformations induced by heat or solvent. © 1993 John Wiley & Sons, Inc.     

Reassessment of the random coil conformation: vibrational CD study of proline oligopeptides and related polypeptides.

The "random coil" conformational problem is examined by comparison of vibrational CD (VCD) spectra of various polypeptide model systems with that of proline oligomers [(Pro)n] and poly(L-proline). VCD, ir and uv CD spectra of blocked L-proline oligopeptides [(Pro)n, n = 2-12] in different solvents are reported and compared to the spectra of poly(L-proline) II, poly(L-glutamic acid), and unblocked proline oligomers. Based on the chain-length dependence of the VCD and electronic CD (ECD) spectra of proline oligomers, it is established that VCD spectra are dominated by short-range interactions. The VCD of random coil model polypeptides is shown to be identical in shape but smaller in magnitude than poly(L-proline) II and of similar magnitude to that of (Pro)n (n = 3, 4). Based on the spectral evidence, it is concluded that the "random coil" conformation has a large fraction of helical regions, conformationally similar to the left-handed, 3(1) polyproline II helix, as was previously suggested by Krimm and co-workers. This conclusion is further supported by studies of effects of salt (CaCl2, LiBr, LiClO4), temperature (5-75 degrees C), and pH on the VCD spectra of L-proline oligomers, poly(L-proline) II, and poly(L-glutamic acid). These show that, after each of these perturbations, a significant local ordering remains in the oligomers and polymers studied, and that charged polypeptides such as poly(L-glutamic acid) are more flexible than are polyproline or even L-proline oligomers.     

Characterization of two sequential polytripeptides containing glycine and ethyl-glutamate residues

The polytripeptides poly(glu(OEt)-gly-gly) and poly(glu(OEt)-glu(OEt)-gly) have been synthesized and characterized using infrared linear dichroism, ultraviolet circular dichroism spectroscopy, and electron microscopy. Both polymers have been obtained in a cross β conformation with antiparallel chains, however, there appear to be significant differences in the stability of this conformation for the two polymers. Poly(glu(OEt)-gly-gly) has also been shown to exist in a 3₁ polyglycine II type helix in aqueous solution whereas films cast from this solution appear to be random. Finally this same polymer can be obtained in either a random or an extended form trifluoroacetic acid depending on the nature of the substrate used for film casting.     

Interfacial behaviour of bovine testis hyaluronidase

The interfacial properties of bovine testicular hyaluronidase were investigated by demonstrating the association of hyaluronidase activity with membranes prepared from bovine testis. Protein adsorption to the air/water interface was investigated using surface pressure-area isotherms. In whichever way the interfacial films were obtained (protein injection or deposition), the hyaluronidase exhibited a significant affinity for the air/water interface. The isotherm obtained 180 min after protein injection into a pH 5.3 subphase was similar to the isotherm obtained after spreading the same amount of protein onto the same subphase, indicating that bovine testicular hyaluronidase molecules adopted a similar arrangement and/or conformation at the interface. Increasing the subphase pH from 5.3 to 8 resulted in changes of the protein isotherms. These modifications, which could correspond to the small pH-induced conformational changes observed by Fourier-transform IR spectroscopy, were discussed in relation to the pH influence on the hyaluronidase activity. Adding hyaluronic acid, the enzyme substrate, to the subphase tested the stability of the interfacial properties of hyaluronidase. The presence of hyaluronic acid in the subphase did not modify the protein adsorption and allowed substrate binding to a preformed film of hyaluronidase at pH 5.3, the optimal pH for the enzyme activity. Such effects of hyaluronic acid were not observed when the subphase was constituted of pure water, a medium where the enzyme activity was negligible. These influences of hyaluronic acid were discussed in relation to the modelled structure of bovine testis hyaluronidase where a hydrophobic region was proposed to be opposite of the catalytic site.     

Adsorption of actin at the air-water interface: a monolayer study

The intrinsic surface activity of the contractile protein actin has been determined from surface tension measurements using the Wilhelmy hanging-plate method. Actin, a very soluble protein, moves from the subphase to the air-water interface to make a film. In the absence of magnesium, actin is monomeric and is known as G-actin. During the compression the monomers change their conformation or orientation at the interface and they are then pushed reversibly into the subphase upon further compression. No collapse occurs. Actin monomers in the presence of magnesium become activated; at concentrations greater than some critical value, actin polymerizes to form filaments of F-actin. The actin filaments have a higher surface activity than the actin monomers either because they are more hydrophobic or because F-actin, a rigid polymer, is much more efficient at creating excluded volume. The actin filaments then form a rigid film at the interface that collapses when the surface area is decreased. At less than the critical concentration, the actin monomers are present in the subphase in their activated form. However, their concentration increases at the interface during film compression until the critical concentration is reached. The surface pressure isotherm in this case has the characteristics of a G-actin film at the beginning of the compression and of an F-actin film at the end of the compression process.     

Protein secondary structure from Fourier transform infrared spectroscopy: a data base analysis

An infrared (ir) method to determine the secondary structure of proteins in solution using the amide I region of the spectrum has been devised. The method is based on the circular dichroism (CD) matrix method for secondary structure analysis given by Compton and Johnson (L. A. Compton and W. C. Johnson, 1986, Anal. Biochem. 155, 155-167). The infrared data matrix was constructed from the normalized Fourier transform infrared spectra from 1700 to 1600 cm-1 of 17 commercially available proteins. The secondary structure matrix was constructed from the X-ray data of the seventeen proteins with secondary structure elements of helix, beta-sheet, beta-turn, and other (random). The CD and ir methods were compared by analyzing the proteins of the CD and ir databases as unknowns. Both methods produce similar results compared to structures obtained by X-ray crystallographic means with the CD slightly better for helix conformation, and the ir slightly better for beta-sheet. The relatively good ir analysis for concanavalin A and alpha-chymotrypsin indicate that the ir method is less affected by the presence of aromatic groups. The concentration of the protein and the cell path length need not be known for the ir analysis since the spectra can be normalized to the total ir intensity in the amide I region. The ir spectra for helix, beta-sheet, beta-turn, and other, as extracted from the data-base, agree with the literature band assignments. The ir data matrix and the inverse matrix necessary to analyze unknown proteins are presented.     

A polycationic amine that induces unique conformational changes in poly(dA-dT) in low salt

Circular dichroism was used to examine alterations in the secondary structure of poly(dA-dT) X poly(dA-dT) upon binding polymer X, a polycationic CD probe for aspects of DNA structure. Stable complex formation is evidenced by increasing Tm and the appearance of large extrinsic bands in the greater than 300 nm, region which increase proportionally with r (ratio of polymer charge to DNa phosphate), in the range 0.0 to 0.32. At relatively low values of r (less than .32), CD spectra of the poly(dA-dT) X poly(dA-dT)-polymer X complex show a gradual non-cooperative inversion in the long wavelength portion (275 nm) of the intrinsic band in low salt solutions suggesting structural and conformational flexibility in poly(dA-dT) X poly(dA-dT) and further implicating polymer X as a potential probe for variations in DNA secondary structure. The dinucleotide repeat configuration of poly(dA-dT) X poly(dA-dT) is presumed to play a role in the observed intrinsic CD changes. NMR data support an "alternating B" conformation for the complex.     

Conformational states of poly(L-tyrosine) in aqueous solution.

Poly(L -tyrosine) [(L -Tyr)_n] has been characterized in aqueous solution using circular dichroism (CD) and infrared (ir) spectroscopy, and ultracentrifugal analysis. Most of the experiments were carried out at 0.01% polymer or less to avoid the complications caused by precipitation previously encountered by others. This permitted us to study solutions of (L -Tyr)_n at lower pH values than had been attained previously. Our results show that a transition to an antiparallel-β conformation occurs at pH 11.32 upon titration from higher pH. The β structure is intramolecular when first formed and aggregates with time or upon titration below pH 11. Ultracentrifugal analysis of the intramolecular β conformation shows that it is quite compact, with a frictional coefficient ratio, f/f_min, of 1.09. In addition to the β structure, a nonordered form of the polymer has been obtained below pH 11 by rapid titration of the ionized polyelectrolyte. This form is nonaggregated and was found to have an f/f_min of 1.01, and is therefore almost spherical. The aggregated β form was found to be thermodynamically more stable than the nonordered form at pH 10.7.     

Vibrational circular dichroism spectra of three conformationally distinct states and an unordered state of poly(L-lysine) in deuterated aqueous solution

Fourier transform ir vibrational circular dichroism (VCD) spectra in the amide I′ region of poly(L-lysine) in D₂O solutions have confirmed the existence of three distinct conformational states and an unordered conformational state in this homopolypeptide. Characteristic VCD spectra are presented for the right-handed α-helix, the antiparallel β-sheet, an extended helix conformation previously referred to as the so-called “random coil,” and a completely unordered conformation characterized by the absence of any amide I′ VCD. VCD for the antiparallel β-sheet in solution and the unordered chain conformation are presented for the first time. Each of the four different VCD spectra is unique in appearance and lends weight to the view that VCD has the potential to become a sensitive new probe of the secondary structure of proteins in solution.     

Circular dichroism of adenine and thymine containing synthetic polynucleotides

Circular dichroism (CD) spectra of poly(dA), poly(dT), poly(dA)·poly(dT), and poly[d(A-T)]·poly[d(T-A)] have been measured as a function of temperature. From these data difference spectra have been calculated by subtracting the spectrum measured at low temperature from the spectra measured at higher temperatures. The CD difference spectra obtained upon melting of the two double-stranded polymers are very similar. From a comparison of these difference spectra with calculated ones it is shown that optical transitions near 272 nm (on A) and 288 nm (most probably on T) are present. The premelting changes of the CD spectrum of poly[d(A-t)]·poly[d(T-A)] are due to a change in conformation in which the secondary structure goes from a C- to B-type spectrum by increasing the A-type nature of the polymer. Such a change is not observed for poly(dA)·poly(dT). Instead, a transition between two different B-type geometries occurs.     

Interaction between polylysine monolayer and DNA at the air-water interface

The interaction of a polylysine amphiphile, which consists of a poly-L- or -D-lysine (1L or 1D) segment and two long alkyl chains at the C-terminus, with polynucleotides was studied with respect to the highly organized structure of polylysine assemblies on water. The results of surface pressure-area isotherm measurement showed that both of 1L and 1D formed stable monolayers on water in a neutral pH region. The secondary structure of polylysine segment for the surface monolayer was examined by means of circular dichroism and Fourier transform infrared spectroscopies. The helical structure was retained even at neutral pH, at which polylysine has been known to form a complete random coiled conformation in bulk solution. Protonated, positively charged and coiled 1L monolayer could interact electrostatically with guest polyanions including DNA in the subphase, and at the same time the conformation of the polylysine segment was converted from a random coil to an alpha-helix. Deprotonated, helical monolayers did not interact with single stranded polyadenylic acid, but with double stranded DNA. Double stranded DNA was found to interact more strongly with right-handed 1L monolayer than left-handed 1D monolayer. An obvious difference in the melting temperatures for these complexes was observed and discussed on the basis of difference in the interaction mode.     

Conformational study of poly(α,β-L-aspartic acid)

The conformation of several samples of poly(α,β-L -Asp) with a molar fraction of β-bonds ranging from 0.1 to 0.55 was investigated by means of ir and CD spectroscopy and potentiometric titration and compared with the results obtained previously with poly(α-L -Asp). All samples investigated underwent a conformational change induced by changes in their degree of ionization: unpronounced ir absorption of amide V at 650 cm^?1 was shifted to 620 cm^?1 and substantially increased on deionization; CD spectra changed with the degree of ionization, passing through an isosbestic point; and the pattern of the titration curves was more complex than that of a simple polyelectrolyte. The conformation developing with the decreasing degree of ionization may be considered to be α-helix, as deduced according to the analogous behavior of other polypeptides. The extent of the conformational change in the individual samples depends on the molar fraction of β-bonds: the higher it is, the lower is the helix-forming ability of the sample.     

Interaction of DNA with poly(L-Lys-L-Ala-Gly) and poly(L-Lys-L-Ala-L-Pro). Circular dichroism and thermal denaturation studies

Complexes of DNA with polypeptides composed of Lys, Ala, and Gly in both a sequential order, poly(L-lysine-L-alanine-glycine), and a statistical distribution, poly(L-lysine36-L-alanine28-glycine), were prepared using gradient dialysis. These polypeptide-DNA complexes were studied using ultraviolet absorption (UV) and circular dichroism (CD) to probe the conformation, binding, and melting behavior of DNA in the complex. Complexes with the sequential polypeptide showed no structural change in the DNA; however, the complexes with the random polypeptide yield CD spectra similar to phi DNA [Maniatis, T., Venable, Jr., J.S., and Lerman, L.S. (1974), J. Mol. Biol. 84, 37]. A second sequential polypeptide, poly(L-Lys-L-Ala-L-Pro)n, -DNA complex was also studied. It was found to exhibit pronounced structural changes as a function of ionic strength and poly-peptide-DNA ratio, more similar to the random sequence that the ordered sequence of the Lys, Ala, Gly polymer. Thus the importance of the composition and amino acid sequence in polypeptides which bind to DNA, even in such simple systems, is demonstrated. Evidence from thermal denaturation, employing simultaneous monitoring of CD and UV changes, supports a model in which specific polypeptides cause condensation of the DNA in the complex into an asymmetric tertiary structure. The relevance of these model systems to chromatin is discussed.     

Partially induced transition from horizontal to vertical orientation of helical peptides at the air–water interface and the structure of their monolayers transferred on the solid substrates

To apply the Langmuir–Blodgett (LB) technique as a platform for investigating the fundamental properties of amphiphilic peptides (APs), we have investigated the structure of LB films using the APs. To vertically orient the helical APs like transmembrane proteins in the membrane, the primary structure of the APs was designed to have two domains: a hydrophilic domain (three amino acids) and a hydrophobic domain (ca. 20 amino acids). However, we are still far from having full control of their orientation. This study reports the contribution of the subphase temperature to the change in the orientation of helical APs. When the surface pressure–area isotherm of AP was observed at the subphase temperature at 41.5 °C, the isotherm exhibited a plateau, implying that a phase transition of the monolayer at the air–water interface occurred. Circular dichroism (CD) spectra of the monolayers transferred on the solid substrates revealed that the orientation of the helices changed at the pressure, where the plateau of the isotherm was observed. This change was not observed at 21.5 °C, i.e., the horizontal alignment of helixes was maintained. Atomic force microscopy (AFM) was used to systematically investigate the surface structure of the monolayers transferred at different surface pressures. A structural model of the monolayer that did not contradict with the results obtained by the three different techniques (the isotherm, CD spectroscopy, and AFM) was derived, and it was concluded that the horizontally oriented helices partially changed their orientation to vertical upon compression in the plateau region of the isotherm.     

Structural composition of betaI- and betaII-proteins

Circular dichroism spectra of proteins are sensitive to protein secondary structure. The CD spectra of alpha-rich proteins are similar to those of model alpha-helices, but beta-rich proteins exhibit CD spectra that are reminiscent of CD spectra of either model beta-sheets or unordered polypeptides. The existence of these two types of CD spectra for beta-rich proteins form the basis for their classification as betaI- and betaII-proteins. Although the conformation of beta-sheets is largely responsible for the CD spectra of betaI-proteins, the source of betaII-protein CD, which resembles that of unordered polypeptides, is not completely understood. The CD spectra of unordered polypeptides are similar to that of the poly(Pro)II helix, and the poly(Pro)II-type (P2) structure forms a significant fraction of the unordered conformation in globular proteins. We have compared the beta-sheet and P2 structure contents in beta-rich proteins to understand the origin of betaII-protein CD. We find that betaII-proteins have a ratio of P2 to beta-sheet content greater than 0.4, whereas for betaI-proteins this ratio is less than 0.4. The beta-sheet content in betaI-proteins is generally higher than that in betaII-proteins. The origin of two classes of CD spectra for beta-rich proteins appears to lie in their relative beta-sheet and P2 structure contents.     

Synthesis of poly(L-Tyr-L-Glu-L-Ala-Gly) and poly(L-Glu-L-Lys-L-Ala) and their circular dichroism spectra in aqueous solution

Two sequential polypeptides, poly(O-benzyl-L -Tyr-γ-benzyl-L -Glu-L -Ala-Gly) and poly(ε-benzyloxycarbonyl-L -Lys-L -Glu-L -Ala), were synthesized, the former by the pentachlorophenyl ester of the tetrapeptide monomer and the latter by the azide of the tripeptide monomer. After deprotection and dialysis, poly(L -Tyr-L -Glu-L -Ala-Gly) was obtained in 71% yield and had a molecular weight of 53,000. The circular dichroism spectra (CD) of the polymer at pH's 7.2, 10.5, and 11.8 and of oligomers and of the monomer at pH 7.2 indicated that the polymer exists in an α-helical conformation. After deprotection, poly(L -Lys-L -Glu-L -Ala) was obtained in 37% yield and had a molecular weight of 3000. The CD spectra of the polymer at pH 7.2 and 2.8, and of the monomer at pH 7.2, indicated that the polymer is in a randomly coiled configuration.     

CD of the synthetic RNA duplexes poly[r(A-T)] and poly[r(A-U)] in salt and ethanolic solutions.

Synthetic RNA poly[r(A-T)] has been synthesized and its CD spectral properties compared to those of poly[r(A-U)], poly[d(A-T)], and poly[d(A-U)] in various salt and ethanolic solutions. The CD spectra of poly[r(A-T)] in an aqueous buffer and of poly[d(A-T)] in 70.8% v/v ethanol are very similar, suggesting that they both adopt the same A conformation. On the other hand, the CD spectra of poly[r(A-T)] and of poly[r(A-U)] differ in aqueous, and even more so in ethanolic, solutions. We have recently observed a two-state salt-induced isomerization of poly[r(A-U)] into chiral condensates, perhaps of Z-RNA [M. Vorlícková, J. Kypr, and T. M. Jovin, (1988) Biopolymers 27, 351-354]. It is shown here that poly[r(A-T)] does not undergo this isomerization. Both the changes in secondary structure and tendency to aggregation are different for poly[r(A-T)] and poly[r(A-U)] in aqueous salt solutions. In most cases, the CD spectrum of poly[r(A-U)] shows little modification of its CD spectrum unless the polymer denatures or aggregates, whereas poly[r(A-T)] displays noncooperative alterations in its CD spectrum and a reduced tendency to aggregation. At high NaCl concentrations, poly[r(A-T)] and poly[r(A-U)] condense into psi(-) and psi(+) structures, respectively, indicating that the type of aggregation is dictated by the polynucleotide chemical structure and the corresponding differences in conformational properties.     

Experimental study of the conformation of two stereoregular polymethionines: poly(D-methionyl-L-methionine) and poly(L-methionyl-D-methionyl-L-methionine) (author's transl)]

Infrared spectroscopy, X-ray diffraction, and nuclear magnetic resonance spectroscopy have been used in investigating the conformation of two stereoregular polymethionines, poly(D -methionyl-L -methionine) and poly(L -methionyl-D -methionyl-L -methionine). When dissolved in a helicogenic solvent, such as chloroform and hexafluoroisopropanol, the polytripeptide is in an α-helical conformation. A helix-to-coil transition can then be induced by addition of trifluoroacetic acid. On the other hand, it appears that the most stable conformation of poly(D -Met-L -Met) is a β antiparallel folded structure in which the linear polypeptide segments are near to the planar extension. This structure has been evidenced through X-ray examination of oriented films, casted from solutions in chloroform. It has also been identified in solution in the same solvent, by use of infrared spectroscopy and by measuring the δ_Hα chemical shift which characterizes the H^α proton in the peptide units. This δ_Hα value is found equal to 5.4 ppm and differs significantly from those which are usually attributed to the α-helical conformation (δ_Hα = 4.2 ppm) and to the random coil (δ_Hα = 4.6 ppm). The β folded conformation of the poly(D -Met-L -Met) appears to be comparatively less stable than the α-helical one for the poly(L -Met) macromolecular stereoisomer since hexafluoroisopropanol is a helicogenic solvent for this last solute and a destabilizing one for the poly(D -Met-L -Met) β folded conformer. X-ray examinations carried out with stretched films, casted from a solution of poly(D -Met-L -Met) in chloroform, result in several data concering the cross β structure of this stereoregular polypeptide in the solid state.     

Secondary structure of polypeptide hormones of the anterior lobe of the pituitary gland

The specific secondary structure of a number of polypeptide hormones of the pituitary gland anterior lobe and their fragments were studied by CD in the peptide bond absorption region and by ir spectroscopy. The state of objects was examined in solvents of different polarity over a wide temperature range as well as in the solid state at different relative humidities. The predominant conformational state of a number of hormones in aqueous solution is shown to represent a left-handed helix of the poly(L -proline) II type. The reversible melting process of the left-handed helical conformation when heated in an aqueous solution appears to be noncooperative. Lowering the temperature stabilizes the left-handed structure. The transition mode of the left-handed form to the α-, and the β-forms on changing the solvent conditions was also studied. Contributions of peptide chromophores and of the aromatic amino acid side-group chromophores with CD bands in the region under study were determined by analysis of CD spectra. The data obtained allow correlating the conformation of separate fragments in the hormone chain with functional activity.