Structural study of poly(beta-benzyl-L-aspartate) monolayers at air-liquid interfaces.

As normally studied, in the solid state or in solution, poly(beta-benzyl-L-aspartate) (PBLA) differs from the other helical polyamino acids in that its alpha-helical conformation is most stable in the left-handed rather than in the right-handed form. The slightly lower energy per residue for the left-handed form in PBLA is easily perturbed, however. The helical screw sense can be inverted in a polar environment and, upon heating above 100 degrees C, a distorted left-handed helix or omega-helix is irreversibly formed. From external reflectance Fourier transform infrared measurements at the air-water interface, the conformation of PBLA in the monolayer state is directly established for the first time. The infrared frequencies of the amide bands suggest that right-handed alpha-helices are formed on the surface of water immediately after spreading the monolayers and independently of the polypeptide conformational distribution in the spreading solution. The right-handed helical form prevails throughout the slow compression of the Langmuir monolayers to collapsed films. The helical screw sense can be reversed by lowering the polarity of the aqueous phase. In addition, an alternate conformation similar to the omega-helix forms on addition of small amounts of isopropanol to the aqueous subphase, and appears to be an intermediate in the helix-helix transition.     

Diffusional behavior of poly(beta-benzyl L-aspartate) in the rodlike, random-coil, and intermediate forms as studied by high field-gradient 1H NMR spectroscopy

The diffusion coefficients of poly(beta-benzyl L-aspartate)(PBLA) with the alpha-helix (rodlike) form in chloroform and the random-coil form in a mixture of chloroform and trifluoroacetic acid have been measured as a function of the PBLA concentration (C(PBLA)) by using high field-gradient 1H NMR. The PBLA concentration range for the former is from 0.11 to 9.20 wt % and for the latter it is from 0.20 to 25.2 wt %. From these experimental results, it is found that the diffusion coefficient of PBLA in the rodlike form is much smaller than that of PBLA in the random-coil form at the same PBLA concentration. This implies that the small diffusion coefficients of PBLA in the rodlike form as compared with those in the random-coil form come from the relatively large radius of gyration Rg. Further, it is found that the diffusional behavior of PBLA in the rodlike form is different in the three PBLA concentration regions, that is, region 1 (C(PBLA) = 0.11 approximately 0.39 wt %), region 2 (C(PBLA) = 0.39 approximately 0.86 wt %), and region 3 (C(PBLA) = 0.86 approximately 9.20 wt %). In the random-coil form, the diffusion is different in the two PBLA concentration regions, that is, region I (C(PBLA) = 0.20 approximately 1.03 wt %) and region II (C(PBLA) = 1.03 approximately 25.2 wt %). These diffusional behaviors in the rodlike form and in the random-coil form can be reasonably explained by Tinland-Maret-Rinaudo theory and de Gennes theory, respectively. Further, transitional change from the rodlike form to the random-coil form is discussed on the basis of the diffusional behavior.     

Ultrasonic absorption studies of poly-benzyl-L-aspartate in mixed solvents, in relation to the helix-cell transition

Ultrasonic absorption measurements were carried out on solutions of polybenzyl-L -aspartate (PBLA) in chloroform–dichloroacetic acid (DCA) and in 1,2-dichloroethane (DCE)–DCA, in the range 3.9–155 MHZ . The helix–coil transition of PBLA produces an increase of absorption which is larger in CHCl₃–DCA than in DCE–DCA solutions. The influence of the solvent on the excess ultrasonic absorption suggests that solvation processes may be involved in these changes of absorption. The plots of the absorption vs. the volume fraction of DCA do not show any absorption maximum. This indicates that the ultrasonic absorption is not sensitive to the helix–coil equilibrium of PBLA in the frequency range investigated. A maximum value of 10⁹ S ^?1 has been obtained for the rate constant of growth of a helix region.     

Structure of aggregates and helix-coil transition of polypeptides by dielectric measurements. II. Poly-D,L-phenylalanine and poly-beta-benzyl-L-aspartate in chloroform-dichloroacetic acid mixtures

The dielectric absorption of poly-DL -phenylalanine and poly-γ-benzyl-L -aspartate (PLAB) was measured in very dilute solutions to determine the type of molecular association and to locate the helix–coil transition. Both polypeptides were present as associated helices in chloroform. The mode of aggregation, which was determined by measuring the dipole moment and the critical frequency, did not depend on the polarity of the side chain but rather on that of the solvent. In both polymers, the dissociation of the aggregates in chloroform was observed on addition of small amounts of dichloroacetic acid; further addition of the acid lead to the helix–coil transition. No second region of dielectric absorption that might be related to the kinetics of the transition was found during the helix–coil transition of PBLA.     

Enthalpy change of the coil-helix transition of poly(gamma-benzyl L-glutamate) in dichloroacetic acid-1,2-dichloroethane mixtures

By combining the heat of dilution of a poly(γ-benzyl L -glutamate)–dichloroacetic acid–1, 2-dichloroethane solution with the corresponding heat of mixing of two solvents, the integrated heat of the coil–helix transition of poly(γ-benzyl L -glutamate) in the solution was estimated to be about 750 cal/mole.     

Solution properties of synthetic polypeptides. V. Helix–coil transition in poly(β-benzyl L-aspartate)

Simple approximate expressions have been derived from the theory of Zimm and Bragg for use in the analysis of experimental data on the helix-coil transition in polypeptide. On the basis of the resulting expressions practical procedures are proposed to determine two basic parameters characterizing a thermally induced transition, i.e., helix initiation parameter σ and enthalpy change for helix formation, ΔH. They have been applied to the data for poly(β-benzyl L -aspartate) (PBLA) with the result: σ = 1.6 × 10^?4 and ΔH = ?450 cal/mole for PBLA in m-cresol; σ = 0.6 × 10^?4 and ΔH = 260 cal/mole for PBLA in chloroform containing 5.7 vol-% of dichloroacetic acid. This result gives evidence that σ may change not only from one polypeptide to another but also for a given polypeptide in different solvents. The change in limiting viscosity number [η] accompanying the transition was measured in the same solvents. The curve of [η] versus helical content had a relatively monotonic shape for the chloroformdichloroacetic acid solutions as compared with that for the m-cresol solutions, indicating that [η] depended largely on σ. Provided that [η] is a direct measure of the mean-square radius of gyration, 〈S²〉, the results are consistent with the theoretical predictions of Nagai and of Miller and Flory for 〈S²〉.     

Adhesion behavior of peritoneal cells on the surface of self-assembled triblock copolymer hydrogels

Adhesion behavior of cells to the surface of physical hydrogel membranes prepared by water-induced self-organization of precisely synthesized ABA-triblock copolymers comprised of poly(beta-benzyl L-aspartate) (PBLA) as A segment and poly(ethylene oxide) (PEO, molecular weight = 20 000) as the B segment were investigated. The cast film from the methylenechloride solution of these copolymers swelled in water very rapidly forming hydrogels (100-400% water content of total weight). The content of PBLA affected the strength, the hydrophobicity, and the amount of water involved in the hydrogel surface. During the early stage of cultivation with murine peritoneal cells, cell adhesion on the hydrogels of PEO and PBLA with 18 (20K18) and 25 (20K25) monomeric units was not observed, while adhesion on the hydrogels of PEO and PBLA with 32 (20K32) and 55 (20K55) monomeric units was successful, suggesting more than 12 mol % in PBLA content is necessary for adhesion of these cells. Although cell spreading on the hydrogels of 20K18, 20K25, and 20K32 was not sufficient, the hydrogel of 20K55 allowed cell adhesion and spreading to be bipolar with leading edge whose raffling is active with pseudopodium and lamellipodium as well as PBLA homopolymer, suggesting active motility of these cells. Remarkably, prolonged incubation restored adhesiveness onto the films at 20K18 in contrast to adhesion with 20K25 despite low hydrophobicity. It is conceivable that adaptation of proteins and chemical changes to the surface during the culture period may participate in these phenomena. Mechanical properties and interaction between cell and these copolymer hydrogels could be controlled by composition of block segments, and optimization for implants could also be attainable.     

Molecular theory of the helix–coil transition in polyamino acids. V. Explanation of the different conformational behavior of valine,isoleucine, and leucine in aqueous solution

Even though poly(L -valine) and poly(L -isoleucine) both contain residues that are branched at their β-carbon atoms, they exhibit a different behavior of their Zimm-Bragg helix-growth parameter s in aqueous solution. This quantity increases with temperature for poly(L -valine) but decreases for poly(L -isoleucine). The origin of this behavioral difference was examined by computing theoretical values of s versus temperature from interatomic interaction energies, taking solvent (hydrophobic and hydrophilic) effects into account. The calculated s versus temperature curves for both homopolymers are consistent with the observed experimental behavior. The two homopolymers behave differently because of differences in the change in the number of hydration-shell water molecules accompanying their helix–coil transitions. The larger isoleucine side chains are more crowded together in both the α-helical and coil forms than are those of valine. Therefore, there is a smaller change in hydration of the isoleucine side chains compared to that of the valine side chains in the helix–coil transition. By analyzing the effects of hydration on the s versus temperature curves, it is possible to account also for the experimental curve for poly(L -leucine), which exhibits an intermediate behavior between those for poly(L -valine) and poly(L -isoleucine).     

Helix-coil transition in copolypeptides. I. Poly (gamma-benzyl-co-gamma-methyl L-glutamate)

The helix–coil transition in poly(γ-benzyl L -glutamate-co-γ-methyl L -glutamate) copolypeptides was studied experimentally in nonaqueous solvents and the results compared with theory. It is found that the transition can be described by the same theory as for the homopolypeptides, but the corresponding parameters are not related in the expected way to those of the homopolymers, due to the effect of the side chains on the stability of the helix, which is not taken into account explicitly by the theory.     

Origin of multipeak behavior in the NMR spectra of poly(L-glutamic acid)

The previous report that poly(L -glutamic acid) exhibits doubled resonances in the helix–coil transition region by either proton or carbon-13 nmr resolves the question of whether or not this behavior is limited to uncharged polypeptides in organic solvents, as had been previously thought. In the present work, we show that the underlying principle causing this anomalous double-peak behavior is due to molecular-weight polydispersity of the sample. The molecular-weight range in which this phenomenon is observed is largely dependent on the values of σ, the nucleation or cooperativity factor. The principles developed are shown to encompass all classes of polypeptides in a very natural way and to explain the key experimental data in the literature.     

Synthesis and conformational study of poly(N delta-carbobenzoxy, N delta-benzyl-L-ornithine) and poly(N delta-benzyl-L-ornithine)

Poly(N^δ-carbobenzoxy, N^δ-benzyl-L -ornithine) (PCBLO) was prepared by the standard NCA method. PCBLO was converted into poly(N^δ-benzyl-L -ornithine) (PBLO) through decarbobenzoxylation with hydrogen bromide. The monomer N^δ-benzyl-L -ornithine was synthesized by reacting L -ornithine with benzaldehyde, followed by hydrogenation. The conformation of the two polypeptides was studied by optical rotatory dispersion and circular dichroism. PCBLO forms a right-handed helix in helix-promoting solvents. In mixed solvents of chloroform and dichloroacetic acid (DCA) it undergoes a sharp helix–coil transition at 12% (v/v) DCA at 25°C, as compared with 36% for poly(N^δ-carbobenzoxy-L -ornithine) (PCLO). Like PCLO, the helix–coil transition is “inverse,” that is, high temperature favors the helical form. PBLO is soluble in water at pH below 7 and has a “coiled” conformation. In 88% (v/v) 1-propanol above pH (apparent) 9.6 it is completely helical. In 50% 1-propanol the transition pH (apparent) is about 7.4; this compares with a pH_tr of about 10 for poly-L -ornithine in the same solvent.     

Conformational studies of polymers and copolymers of L-aspartate esters. I. Preparation and solution studies

An alcoholysis method is described for the modification of high molecular weight poly(β-benzyl L -asparatate); by this method the benzyl groups in the polypeptide have been replaced by methyl, ethyl, isopropyl, n-propyl, and phenethyl groups to give a series of copolymers of each of the corresponding aspartate esters with benzyl L -aspartate. By repeating the reactions, replacement of better than 99% has been achieved in some cases to give in effect the homopolymer. Optical rotatory dispersion studies show that of all the systems studied only poly(β-methyl L -aspartate) has the left-handed helix sense, the others are right-handed. It is shown further that the helix sense is not an intrinsic property of the nature of the aspartate side chain. Raising the temperature of chloroform solutions of the right-handed form of the copolymers of benzyl L -aspartate and ethyl L -aspartate results in a transition to the left-handed helix, the temperature of the transition being dependent on the composition of the copolymer. Also poly(β-n-propyl L -aspartate) undergoes a transition from the right- to the left-handed helix form at 59°C. These results suggest a general pattern of behavior of poly(aspartate esters) and that with suitable conditions of solvent and temperature they may be in either the right- or left-handed helical form.     

Rotatory dispersion and circular dichroism of brain "proteolipid" protein

Abstract— The proteolipid and its partially delipidated protein from brain tissue have been studied by optical rotatory dispersion and circular dichroism in various solvents. The protein exhibits a helix content of 60–70 per cent in chloroform-methanol and 2-chloroethanol and is dextrorotatory in the visible region. In 1,1,1-trinuoroethanol, a higher helix content of 90 per cent is observed but the protein is laevorotatory. The helix content decreases in water and methanol with a much greater negative specific rotation in methanol. No random coil form has been observed.     

Structure-function analysis of the C-terminal domain of LcrV from Yersinia pestis

LcrV, a multifunctional protein, acts as a positive regulator of effector protein secretion for the type III secretion system (T3SS) in Yersinia pestis by interaction with the negative regulator LcrG. In this study, LcrV was analyzed to identify regions required for LcrG interaction. Random-linker insertion mutagenesis, deletion analysis, and site-directed mutagenesis of hydrophobic amino acids between residues 290 and 311 allowed the isolation of an LcrV mutant (LcrV L291R F308R) defective for LcrG interaction. The new residues identified in LcrG interaction lie in helix 12 of LcrV; residues in helix 7 of LcrV are known to be involved in LcrG interaction. Helix 7 and helix 12 of LcrV interact to form an intramolecular coiled coil; these new results suggest that the intramolecular coiled coil in LcrV is required for LcrG interaction and activation of the T3SS.     

Conformational studies of polymers and copolymers of L-aspartate ester. II. Infrared studies and the factors involved in the formation of the omega-helix

It has already been show that the helix senses of poly(β-benzyl L -aspartate) and poly(β-methyl L -aspartate) are left-handed, while the poly esters of n-propyl, isopropyl, n-butyl, and phenethyl L -asparate are all right-handed. The effect of changes in helix sense from the left-handed to the right-handed α-helical form on the infrared spectra of copolymers of benzyl L -aspartate with ethyl, n-butyl, isopropyl, n-propyl, and phenethyl L -aspartate have been studied. Those show that for the right-handed helical form the amide band frequencies fall within the range given by Elliott,⁷ while for the left-handed form the frequencies are higher. The frequency ranges for the two helix senses are given and have been used to show that poly (β-n-propyl L -aspartate) in chloroform solution undergoes a transition from the right-handed to the left-handed helix form on heating. Polarized infrared studies of the different copolymers show that the disposition of the side chain ester groups is different for the two forms. Although methyl L -aspartate forms a left-handed α-helix similar to benzyl L -aspartate, the introduction of methyl L -aspartate residues into poly (β-benzyl L -aspartate) prevents the formation of the ω-helix. The factors involved in the formation of this helix form are discussed.     

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.     

Reversal in helix sense of copoly(beta-alkyl-L-aspartate-beta-benzyl-L-aspartate)

The conformation of copoly(beta-alkyl-L-aspartate-beta-benzyl-L-aspartate), in which the alkyl group is ethyl, propyl, butyl, hexyl, nonyl, dodecyl, or stearyl, was studied in solution and the solid state by optical rotatory dispersion and circular dichroism methods. The helix sense of the copolyaspartate studied here is transformed from a left-handed to right-handed alpha-helix as the degree of alkylation increases. Reversal in helix sense occurs, i.e., the left-handed alpha-helix based on the handedness of poly(beta-benzyl-L-aspartate) is transformed into a right-handed alpha-helix with increase in alkyl groups with right-handed nature. Reversal in helix sense is also observed for copolyaspartates with an intermediate or high degree of alkylation as temperature rises. Copolyaspartates with hexyl, nonyl, or dodecyl groups exhibit an induced circular dichroism around 230-238 nm and can form an ordered side chain structure which is broken down at high temperature. One has to consider the conformation of the omega-helix and beta-form of the copolyaspartates in the solid state in addition to the reversal in helix sense. Copolyaspartates with a low degree of alkylation are in the alpha-helical conformation over the low temperature range and adopt the omega-helical conformation in the high temperature range, indicative of a thermal alpha-omega transition. A small number of alkyl groups can be incorporated into the benzene ring stacking of the omega-helix, but not a large number. All the copolyaspartates can assume the beta-form at high temperatures. The helix conformation is not significantly affected by the formation of side chain crystals of the copolyaspartate with a large number of stearyl groups, in contrast to copolyglutamate.     

Carbon-13 and proton NMR studies of helix-coil transition of poly(gamma-benzyl-L-glutamate).

The helix–coil conformational transition undergone by poly(γ-benzyl-L -glutamate) in solutions of trifluoroacetic acid and deuterated chloroform was studied by proton and carbon-13 nmr. The results indicate that in the case of the solvent-induced helix–coil transition, the side chain assumes a helical conformation before the backbone. In the thermally induced helix–coil transition, the results indicate the existence of an intermediate state, which is between the α-helix and random coil and is free from intramolecular hydrogen bonding.     

Disorder–order transitions induced in anionic homopolypeptides by cationic detergents

CD spectra have been obtained for poly(L -glutamic acid) and poly(L -aspartic acid) as functions of temperature and concentration of cationic detergents. Dodecylammonium chloride induces a coil–helix transition in fully ionized poly(L -glutamic acid). The interaction of the monomeric detergent with the polypeptide is responsible for the conformational transition. The detergent concentration required to produce the transition is independent of temperature. The CD of fully ionized poly(L -aspartic acid) is nearly unaffected by dodecylammonium chloride, in marked contrast to the situation found with poly(L -glutamic acid). However, these results do not imply that dodecylammonium chloride interacts differently with aspartyl and glutamyl residues. The observed results can be accounted for by the well-known fact that the glutamyl residue has a higher helix-forming tendency that the aspartyl residue. Cetyltrimethylammonium chloride destabilizes the helical form of poly(L -glutamic acid). This detergent presents an exception to the usual ability of ionic detergents to promote formation of ordered structures in oppositely charged homopolypeptides.     

Activation of the cannabinoid CB1 receptor may involve a W6 48/F3 36 rotamer toggle switch.

The cannabinoid CB1 receptor, a member of the Rhodopsin (Rho) family of G protein coupled receptors (GPCRs), exhibits high levels of constitutive activity. In contrast, Rho exhibits an exquisite lack of constitutive activity. In Rho, W6.48(265) on transmembrane helix 6 (TMH6) is flanked by aromatic residues at positions i-4 (F6.44) and i + 3 (Y6.51), while in CB1 the residues i-4 and i + 3 to W6.48 are leucines (L6.44 and L6.51). Based upon spectroscopic evidence, W6.48 has been proposed to undergo a rotamer switch (chi1 g+ -->trans) upon activation of Rho. In the work reported here, the biased Monte Carlo method, Conformational Memories (CM) was used to test the hypothesis that the high constitutive activity exhibited by CB1 may be due, in part, to the lack of aromatic residues i-4 and i + 3 from W6.48. In this work, the W6.48 rotamer shift (chi1 g+ -->trans) was used as the criterion for activation. Conformational Memories (CM) calculations on WT CB1 TMH6 and L6.44F and L6.51Y mutant TMH6s revealed that an aromatic residue at 6.44 tends to disfavor the W6.48 chi1 g+ -->trans transition and an aromatic residue at 6.51 would require a concomitant movement of the Y6.51 chi1 from trans-->g+ when the W6.48 chi1 undergoes a g+ -->trans shift. In contrast, CM calculations on WT CB1 TMH6 revealed that the presence of leucines at 6.44 and 6.51 provide W6.48 with greater conformational mobility, with a W6.48 transchi1 preferred. Conformational Memories calculations also revealed that the W6.48 chi1 g+ -->trans transition in WT CB1 TMH6 is correlated with the degree of kinking in TMH6. The average proline kink angles for TMH6 were higher for helices with a W6.48 g+ chi1 than for those with a W6.48 transchi1. These results are consistent with experimental evidence that TMH6 straightens during activation. Transmembrane helix (TMH) bundle models of the inactive (R) and active (R*) states of CB1 were then probed for interactions that may constrain W6.48 in the inactive state of CB1. These studies revealed that F3.36 (transchi1) helps to constrain W6.48 in a g+ chi1 in the inactive (R) state of CB1. In the R* state, these studies suggest that F3.36 must assume a g+ chi1 in order to allow W6.48 to shift to a transchi1. These results suggest that the W6.48/F3.36 interaction may act as the 'toggle switch' for CB1 activation, with W6.48 chi1 g+/F3.36 chi1 trans representing the inactive (R) and W6.48 chi1 trans/F3.36 chi1 g+ representing the active (R*) state of CB1.