Surfactant bound polypeptides: 1. Conformation in solution and solid state of poly (γ-benzyl-l-glutamate) — Triton X-100

N-carboxyanhydride of γ-benzyl-l-glutamate was polymerized using the potassium salt of the non-ionic surfactant Triton X-100, octylphenoxypolyethoxyethanol, as the initiator. N.m.r. spectra revealed the presence of the initiator moiety in the polymer. The surfactant bound polypeptides with a degree of polymerization of 21 and 26 had excellent solubility in several non-aqueous solvents as compared to the homopolypeptide poly-γ-benzyl-l-glutamate (PBLG). The circular dichroic profiles of the polymers in the helicogenic solvents trifluoroethanol and trimethyl phosphate were helical. I.r. spectra of films of the polymers also indicated α-helical conformation for the peptide block. X-ray diffraction data, on the other hand, showed along with a spacing corresponding to the hexagonal packing of helices, a 22 Å spacing, indicative of a new kind of long range order not observed with the homopoly-γ-benzyl-l-glutamate. Again, the polymers formed with facility mesophases in various solvents at concentrations, unlike PBLG, as low as 5–8% (w/v).     

On the double peak of NMR in the helix—coil transition region

To attempt to resolve the controversy over “fast” and “slow” helix–coil transition rates in polypeptides, nuclear magnetic resonance spectra were measured for monodisperse poly-γ-benzyl-L -glutamate (PBLG). These results were compared with simulated line spectra which were computed by taking the molecular-weight distribution into consideration. Broad but single peaks have been observed in 220 mHz nmr for the α-CH and NH proton resonance spectra in the transition region. The shape of the line changes with the extent of polydispersity. Assuming a fast conversion rate, a molecular model of the helix–coil transition simulates these results. Consequently, the double peak which has been observed in the nmr of polypeptides at the helix–coil transition region is shown to result from the polydispersity in molecular weight.     

Solution properties of synthetic polypeptides. XVII. Stability of the alpha-helical conformation of poly (gamma-benzyl L-glutamate) in helicogenic solvents

The Zimm–Bragg parameters s and σ were determined for poly(γ-benzyl L -glutamate) (PBLG) in m-cresol and in dimethylformamide (DMF) from ORD data as a function of molecular weight. It was found that, within the temperature range between 10 and 55°C and on the average, s = 1.6₁ ± 0.1 and √σ = 0.04 ± 0.01 in m-cresol and s = 1.6₅ ± 0.05 and √σ = 0.045 ± 0.015 in DMF. The values of s in m-cresol decreased with increasing temperature, while the values of σ in the same solvent increased. This result for s suggests that PBLG in m-cresol will undergo a thermal helix–coil transition of normal type. The parameters in DMF showed no appreciable trend to vary with temperature. Aside from the difference between the two solvents, our results are consistent with existing data for various conformation-dependent properties such as light-scattering radius, intrinsic viscosity, and dipole moment, each indicating that the polypeptide chain has some flexibility in helicogenic solvents.     

Proof of the charging of polypeptides and polyamides in organic acids

The electrophoresis of poly-γ-benzyl-L -glutamate (PBLG), poly-L -alanine (PLA) and nylon dissolved in various solvents was studied in a glass cell containing three sintered glass partitions. After the passage of a measured amount of charge the concentration of PBLG remained constant in all four chambers when the helicogenic solvents dimethylformamide and ethylene dichloride were used, but in mixtures of ethylene dichloride and dichloroacetic acid and in trifluoroacetic acid, polypeptide migrated to the cathode. Electrophoresis also occurred with PLA in trifluoroacetic acid and with nylon in formic acid. Although the total charge on the polyion could not be determined, the results show beyond reasonable doubt that polypeptides and polyamides are protonated in the presence of moderately strong organic acids.     

Physicochemical study of trisequence copolymers, poly(gamma-benzyl-L-glutamate)-poly(ethylene oxide)-poly(gamma-benzyl-L-glutamate). Role of polydispersity in the determination of conformation of the molecules in dilute solution

We have prepared a series of three-block copolymers containing the same central block of poly(ethylene oxide) (POE) and two side blocks poly(γ-L -glutamate) (PBLG) of variable lengths. These molecules could present an unusual conformational model when dissolved in solvents where PBLG exhibits a helical structure: a helical structure: a gaussian chain connected at each end to a rigid rod. We checked this hypothesis by light scattering, viscosimetry, and dielectric adsorption measurements. In order to take into account the possibility that the orientations of the two rods may not be completely independent, we have introduced in the calculations the angle θ, formed by the two rigid segments. We have mainly considered three types of conformation which can be described by assimilating the lods of PBLG of two vectors having their origin at the juncture with the POE chain: depending whether cos θ (the average value of cos θ) is equals to ?1,0, or +1 ( where the two vectors are antiparalles, oriented independently, or parallel, respectively). We have tried to establish a theoretical expression for the molecular size obtained by each method of investigation, as a function of cos θ . We corrected this value to account for the effect of the polydispersity on the molecular size and then compared the theoretical values thus calculated with the experimental results. The measurements of the radius of gyration, R, by light scattering does not permit the unambiguous determination of cos θ . As soon as the system is not perfectly monodisperse, the variation of R as a funciton of cos θ is not very large. The viscosity results are also difficult to interpret because the effect of the polydispersity on the viscosity cannot be established fo each conformational model. However, a clear answer can be obtained by the dielectric absorption technique for two reasons: the variation of the dielectric absorption as a function of cos θ is very important and the influence of the polydispersity on the measurements is weaker than for the two former methods. The results show that cos θ is equal to ?1, demonstrating that the two vectors are antiparallel. From the dielectric dispersion, one sees that the total length of the molecule is roughly equals to the sum of the lengths of the two rigid blocks. To explain this result we propose a new conformational model. Since it is known that in some solvents and mixtures of solvents the molecules of PBLG tend to associate, we assume that the two rigid rods are antiparallel and partly associated. The associated part of the molecule is assumed to be surrounded by the mixture of POE and solvent, which acts as a poor solvent (ef. Fig. 2.).     

Laser light scattering of PBLG in DMF

We report measurements of static and dynamic properties of poly(γ-benzyl-L -glutamate) (PBLG) in dimethyl formamide at 25 °C using laser light scattering. Correlation-function profile analyses were performed using the histogram method. The resultant average linewidth and dispersion were reconfirmed using the cumulants method. We were able to determine the molecular-weight distribution of PBLG using the histogram method and to confirm the polydispersity for rod polymers in solution to be roughly M_w/M_n ～ 1 + μ₂/Γ ². In our analysis of the Rayleigh linewidth, we used both the rigid-rod model and the Fujime approach involving rod flexibility. We obtained very reasonable agreement between the Fujime theory and our experiments and concluded that for most stiff polymers of interest, it would be more appropriate to use the Fujime approach, which could also yield a rod-flexibility parameter. In semidilute solutions, we observed two effects, namely, a maximum in the diffusion coefficient and a broadening of the linewidth distribution function at both low and high frequencies, which were not predicted by the Doi-Edwards theory.     

Effects of linker length and flexibility on multivalent targeting

Increasing valence can enhance the ability of molecular targeting constructs to bind specifically to targeted cells for drug delivery. Here, we mathematically model the length and flexibility of a linker used to conjoin two peptide ligands of a divalent targeting construct and investigate the influence both on binding avidity and specificity. Four different models are used to approximate varying degrees of linker flexibility (random coil, rigid rod, jointed rods, and combined rod-random coil) and for each linker a binding enhancement factor (VR) is derived that quantifies the increased rate of each construct's second binding event over the first. Results indicate that the moderately flexible models can best reproduce experimentally measured avidities. Also, the magnitude of VR, in conjunction with receptor density and ligand concentration, significantly influences the achievable specificity. Thus, the model elucidates important considerations in designing multivalent targeting constructs for use in delivery of targeted therapy or imaging.     

Solution properties of synthetic polypeptides. II. Sedimentation and viscosity of poly-γ-benzyl-L-glutamate in dimethylformamide

Nine samples of poly-γ-benzyl-L -glutamate (PBLG), ranging in M?_w from 19,000 to 410,000, were examined viscomctrically and by ultracentrifugation with dimethylforma-mide (DMF) at 25°C. as helicogenic solvent. The data for [η] and s₀ (limiting sedimentation coefficient) as functions of M?_w were fitted well by the theories for a rigid prolate ellipsoid of revolution whose major axis increases linearly with M?_w, but whose minor axis is independent of M?_w. This implies that the overall shape of the PBLG molecule in DMF is represented by a straight cylinder whose cross section is independent of its length. The length per monomeric residue h evaluated from [η] is about 1.3 A., whereas that from s₀ is about 1.6 A. No adequate explanation for this difference in h can be found at present. More serious is the fact that these hydrodynamically evaluated values of h are appreciably larger than, the value obtained from our light-scattering measurements reported previously. All these values of h from our studies are not consistent with the value characteristic of the α-helix, for which h is 1.5 A. The concentration dependence of s₀ was found to agree well with the recent theoretical prediction of Peterson for cylindrical macromolecules.     

Infinite-dilution viscoelastic properties of poly-gamma-benzyl-L-glutamate in helicogenic solvents

The storage and loss shear moduli, G, and G?, have been measured for solutions of three samples of poly-γ-benzyl-L -glutamate with molecular weights from 16 to 57 × 10⁴, by use of the Birnboim-Schrag multiple-lumped resonator. The frequency range was 106 to 6060 H_z, the concentration range 0.0015–0.005 g/ml, and the temperature 25°C. Two helicogenic solvents with widely different viscosities, dimethylformamide and m-cresol, were used to provide a broader effective frequency range. The intrinsic moduli, extrapolated to infinite dilution, were compared with the predictions of the theory of Ullman for rigid rods; agreement was rather good at the lowest frequencies, but unsatisfactory at high frequencies. The data over the entire frequency range of three of logarithmic decades could be described closely by a relaxation spectrum consisting of one terminal relaxation time separated by a gap from a sequence of relaxtion times spaced as in the Zimm theory. The terminal time agrees approximately with that calculated for end-over-end rotation of a rigid rod. The additional relaxation mechanisms are tentatively attributed to modes of flexural deformation of the helix.     

Conformational studies on concentrated solutions of poly(gamma-benzyl L-glutamate)

We have studied the effect of polypeptide concentration on the helix–coil transition of poly(γ-benzyl L -glutamate) (PBLG) in both dichloroacetic acid (DCA) and DCA–chloroform (CHF) mixtures. In agreement with other reports, we find the van't Hoff transition enthalpy to be strongly dependent on PBLG concentration. Also, an apparent effect of polypeptide concentration was noted on the transition temperature; however, corrections for finite PBLG concentration on the mole fraction of DCA seem to remove this effect. In order to explain our data, as well as some calorimetric data in the literature, we consider the transition free energy and enthalpy as a sum of three partial terms. These represent the thermodynamic parameters associated with: (1) conformational changes of the polypeptide, e.g. formation or disruption of intramolecular hydrogen bonds; (2) binding by the strong acid to the nonhelical segments of the polypeptide; (3) an overall (weak) interaction of the polypeptide with the nonbound solvent giving rise to dilution parameters that are dependent on the polypeptide conformation. The latter effect is generally ignored, since it is assumed that solvent interactions, other than specific binding, are similar for both the helical and the nonhelical conformation. Striking effects of water (small amounts) and solution aging on the formation of PBLG helices was observed. Water, as expected, acts as a helicogenic solvent when combined with DCA. The processes occurring during solution aging are not known, although the net effect is to stabilize the helical conformation. Finally, we present some rather unique thermally induced transitions of concentrated PBLG (about 200 mg/ml) in DCA. At low temperatures the soluble randomly coiled conformation is present. Heating produces first an isotropic gel, followed at higher temperatures by an isotropic solution consisting of about 70% α-helicity.     

Molecular blending by polymerization of intercalated solvent. Poly(gamma-benzyl-L-glutamate)/benzyl methacrylate as a model system

The aim of the present research is to obtain blending between a polymer and a (polymerized) solvent on the molecular level. Because of its rigid rod structure, poly(gamma-benzyl-L-glutamate) (PBLG) is chosen as the polymer. Benzyl methacrylate (BzMA) has been chosen as the solvent for two reasons. First, the structure of the solvent is very similar to the structure of the side chain of PBLG, favoring interactions between the two materials. Second, the solvent can be polymerized, because of the presence of a C=C bond. In cast films of PBLG and BzMA separate zones of the polymer and solvent are present. Wide-angle X-ray diffraction and Raman results show that upon heating the cast films homogenization occurs and solvent molecules intercalate between the helices of PBLG. At 150 degrees C a hexagonal packing is obtained. The dimensions of the obtained packing depend on the solvent concentration, which confirms that solvent molecules are indeed present within the crystalline lattice. DSC experiments imply that the observed changes upon heating correspond to thermodynamic processes. On cooling the homogeneous samples, disordering of the hexagonal packing occurs. Polymerization of the homogeneous samples results in a disordering of the hexagonal packing and in a contraction of the unit cell. The latter once more confirms that solvent molecules are indeed present within the crystalline lattice. The applied principle of polymerization of a solvent in a molecular homogeneous system can be favorable for many applications, for which morphology control at the molecular level is required.     

Inducible and repressible acetylornithine delta-transaminase in Escherichia coli: different proteins

Two acetylornithine δ-transaminases which have different physical and kinetic properties have been isolated from a mutant of E. coli W. Sephadex gel filtration has shown the molecular weight of one transaminase to be approximately 119,000; the second transaminase has a molecular weight of about 61,000. The two transaminases can be separated by ammonium sulfate fractionation. The K_m values of the smaller and larger molecular-weight species for N^α-acetylornithine are 3.1 mm and 1.3 mm, respectively. The K_m for α-ketoglutarate is 1.1 mm for both enzymes. The presence of arginine in the growth medium represses the synthesis of the 119,000 molecular-weight transaminase and induces the synthesis of the 61,000 molecular-weight species.     

Influence of manufacturing variables on the characteristics and effectiveness of chitosan products. I. Chemical composition,viscosity, and molecular-weight distribution of chitosan products

Ten chitosan products were manufactured from dry shrimp hulls under differing process conditions and compared to a commercially available product. Manufacturing variables tested were: alkali versus enzymatic deproteination; acid demineralization versus no treatment; air versus nitrogen atmosphere; 5 min vs. 15 min deacetylation period: and varying the particle size of the dry starting material. Deproteination by alkali of enzymatic extraction did not substantially affect the nitrogen and ash compositions of dry chitosan samples. However, the viscosity was reduced in samples deproteinated by enzymatic hydrolysis. Elimination of the demineralization step resulted in products having 31–36% ash, as expected. Some differences in viscosity were observed between deminiralized and undemineralized samples, but on important differences in the molecular-weight distribution of these samples were evident. Purging the reaction vessel with nitrogen resulted in chitosan preparations having higher viscosities and molecular-weight distributions than those prepared in an air atmosphere. The degradative effect of air became more proshrimp hulls to 1 mm prior to any treatment resulted in a chitosan product of both higher viscosity and molecular weight than when ground to either 2 or 6.4 mm. Viscosity was not always a direct indicator of molecular weight, for although the presence of colloidal particles increased the viscosity of some samples, the molecular-weight distribution after filtration was essentially the same as in other less viscous samples.     

Surfactant bound polypeptides: 1. Conformation in solution and solid state of poly (γ-benzyl-l-glutamate) — Triton X-100

N-carboxyanhydride of γ-benzyl-l-glutamate was polymerized using the potassium salt of the non-ionic surfactant Triton X-100, octylphenoxypolyethoxyethanol, as the initiator. N.m.r. spectra revealed the presence of the initiator moiety in the polymer. The surfactant bound polypeptides with a degree of polymerization of 21 and 26 had excellent solubility in several non-aqueous solvents as compared to the homopolypeptide poly-γ-benzyl-l-glutamate (PBLG). The circular dichroic profiles of the polymers in the helicogenic solvents trifluoroethanol and trimethyl phosphate were helical. I.r. spectra of films of the polymers also indicated α-helical conformation for the peptide block. X-ray diffraction data, on the other hand, showed along with a spacing corresponding to the hexagonal packing of helices, a 22 Å spacing, indicative of a new kind of long range order not observed with the homopoly-γ-benzyl-l-glutamate. Again, the polymers formed with facility mesophases in various solvents at concentrations, unlike PBLG, as low as 5–8% (w/v).     

Electron paramagnetic resonance analysis of the vimentin tail domain reveals points of order in a largely disordered region and conformational adaptation upon filament assembly

Very little data have been reported that describe the structure of the tail domain of any cytoplasmic intermediate filament (IF) protein. We report here the results of studies using site directed spin labeling and electron paramagnetic resonance (SDSL‐EPR) to explore the structure and dynamics of the tail domain of human vimentin in tetramers (protofilaments) and filaments. The data demonstrate that in contrast to the vimentin head and rod domains, the tail domains are not closely apposed in protofilaments. However, upon assembly into intact IFs, several sites, including positions 445, 446, 451, and 452, the conserved “beta‐site,” become closely apposed, indicating dynamic changes in tail domain structure that accompany filament elongation. No evidence is seen for coiled‐coil structure within the region studied, in either protofilaments or assembled filaments. EPR analysis also establishes that more than half of the tail domain is very flexible in both the assembly intermediate and the intact IF. However, by positioning the spin label at distinct sites, EPR is able to identify both the rod proximal region and sites flanking the beta‐site motif as rigid locations within the tail. The rod proximal region is well assembled at the tetramer stage with only slight changes occurring during filament elongation. In contrast, at the beta site, the polypeptide backbone transitions from flexible in the assembly intermediate to much more rigid in the intact IF. These data support a model in which the distal tail domain structure undergoes significant conformational change during filament elongation and final assembly.     

The amphipathic membrane proteins associated with gangliosides: The Paul-Bunnell antigen is one of the gangliophilic proteins

Two amphipathic protein fractions soluble in organic solvents as well as in water have been isolated from the ganglioside fraction of bovine erythrocyte membranes by successive chromatography in chloroform-methanol mixture on DEAE-Sephadex, silicic acid, and α-hydroxypropylated Sephadex G50 (LH60) columns. These two fractions contained a similar low molecular weight protein but with distinctively different amino acid composition. One of these proteins has been characterized by having a strong Paul-Bunnell antigen activity and had a binding affinity to ganglioside. A similar protein without Paul-Bunnell antigen activity was isolated as the major ganglioside-associated protein.     

Organization of the coiled-coils in the wool microfibril

Two helix-enriched fragments were isolated from a partial tryptic digest of the microfibrillar proteins of wool. From the known sequences of two of the constituent microfibrillar polypeptides, components 7c and 8c-1, one of these fragments was identified as arising from the N-terminal half of the helix-rich region and the other from the C-terminal half of this region. On the basis of the molecular weight in benign and dissociating solvents, crosslinking with dimethylsuberimidate, and amino acid and sequence analyses the N-terminal fragment was shown to be a tetrameric structure, consisting of two segments from each of the components 7 and 8 families. The C-terminal fragment was shown to be a two-stranded coiled-coil containing one chain segment from each of components 7 and 8. The evidence is not compatible with anything but a two-stranded coiled-coil as the basic molecular structure for the wool microfibril and provides experimental proof of a parallel arrangement of the chains. The thermal stabilities of the two fragments and of the parent microfibrillar complex from which they are derived are very similar indicating that the non-helical end regions of the polypeptide chains do not greatly affect the stability of the α-helical rod domain.     

Aggregation of poly(γ-benzyl-α,L-glutamate)

The aggregation of poly(γ-benzyl-α,L -glutamate) and its enantiomer in toluene has been investigated by following the viscosity as a function of temperature, concentration, molecular weight, molecular-weight distribution, helix chirality, and shear rate. The temperature and concentration data for a 138,000-molecular-weight sample was fitted to an open, reversible end-to-end aggregation model. The aggregation numbers resulting from this fit were consistent with the sudden onset in non-Newtonian flow resulting from only a 0.2-wt% increase in concentration. The association equilibrium constant was then used to predict viscosity for comparison with other data, in particular, the effect of molecular weight and molecular-weight distribution. A mixture of right-and left-handed helices showed the aggregation was not chiral selective. The stiffness of end-to-end aggregated (hydrogen-bonded) molecules differed little from their covalent counterparts, at least below a molecular weight of ～10⁶. We conclude that polybenzylglutamate aggregation in toluene can be described by an open end-to-end aggregation model.     

Conformational study on poly [gamma-(alpha-phenethyl)-L-glutamate] using vibrational circular dichroism spectroscopy

Vibrational circular dichroism (VCD) and IR absorption spectra are obtained in a chloroform solution for poly[gamma-((R)-alpha-phenethyl)-L-glutamate] (PRPLG) and poly[gamma-((S)-alpha-phenethyl)-L-glutamate] (PSPLG), whose only structural difference is an opposite chiral center in the side chain. Their characteristic amide A, I, and II bands show VCD patterns quite similar to those of poly[gamma-benzyl-L-glutamate] (PBLG), indicating that the secondary structure of these polypeptides is a right-handed alpha-helix. The VCD spectra in the CH stretching region exhibit different patterns for PRPLG and PSPLG, reflecting the chirality difference in the side chains. This difference is interpreted on the basis of the additivity of optical activity contributions from the main chain conformation and the chirality difference in the side chains. The results indicate that a VCD difference spectrum of the CH stretching region is a useful diagnostic tool for elucidating local chirality differences.