Osmotically induced helix-coil transition in poly(glutamic acid)

Protein folding and conformational changes are influenced by protein-water interactions and, as such, the energetics of protein function are necessarily linked to water activity. Here, we have chosen the helix-coil transition in poly(glutamic acid) as a model system to investigate the importance of hydration to protein structure by using the osmotic stress method combined with circular dichroism spectroscopy. Osmotic stress is applied using poly(ethylene glycol), molecular weight of 400, as the osmolyte. The energetics of the helix-coil transition under applied osmotic stress allows us to calculate the change in the number of preferentially included water molecules per residue accompanying the thermally induced conformational change. We find that osmotic stress raises the helix-coil transition temperature by favoring the more compact α-helical state over the more hydrated coil state. The contribution of other forces to α-helix stability also are explored by varying pH and studying a random copolymer, poly(glutamic acid-r-alanine). In this article, we clearly show the influence of osmotic pressure on the peptide folding equilibrium. Our results suggest that to study protein folding in vitro, the osmotic pressure, in addition to pH and salt concentration, should be controlled to better approximate the crowded environment inside cells.     

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.     

Azobenzene-modified poly(l-glutamic acid) (AZOPLGA): its conformational and photodynamic properties

Azobenzene-modified poly(l-glutamic acid) (AZOPLGA) polymers with 22 and 35 mol % of azo chromophores in the side chains have been synthesized by condensing 4-methoxy-4'-aminoazobenzene and poly(l-glutamic acid). These polymers have been characterized by NMR, FT-IR, and UV-visible spectroscopic techniques. The conformational features of the polymer backbone chains in the films that were cast from the polymer solutions prepared in different solvents have been investigated by circular dichroism spectroscopy. Experimental data suggested that the thermal cis-trans relaxation and photoinduced birefringence, which are related to the azo chromophores in the side chains of polymer, are not affected by the conformations of polymer backbones. However, the modulations of the surface relief gratings, the result of photoinduced mass transport process, recorded on these polymers are sensitive to polymer main chain conformation, as well as the degree of functionalization.     

Potentiometric titrations and the helix-coil transition of poly(L-glutamic acid) and poly-L-lysine in aqueous salt solutions

The objective have been to establish if those ions which are known to change the stability of the structure of proteins, have any influence on the properties of ionizable polypeptides. Potentiometric titrations and complementary optical rotation data are presented for aqueous solutions of poly-L -lysine (PLL) in the presence of KSCN, KCl, and KF, and for poly(L -glutamic acid) (PLGA) in the presence of KSCN, KCl, and LiCl. The following measured quantities which are affected by salt concentration were obtained: intrinsic pK (pK₀), slope of pK_app versus degree of ionization (α) curves, the degree of ionization at which the helix to coil transition occurs, and the free energy of this transition for the uncharged molecule (δG°_hel). The effects of nonspecific salts (KCl and LiCl for PLL and KSCN and KCl for PLGA) are small, and about, as expected from general electrostatic considerations. In line with the observations made with isoelectric and cat ionic collagen, specific, effects were noted with KSCN–PLL and with LiCl–PLGA. In the presence of KSCN, the poly-L -lysine helix becomes stabilized at much lower degree of ionization than in the presence of KCl, and the slope of the pK_app versus α plots is greatly reduced. However, ΔG°_hel (for the uncharged molecule) is not affected, and pK₀ is only slightly higher. We interpret these data in terms of binding of SCN^? primarily to the side-chain amino groups (both to R? NH₃⁺ and to R? NH₂) solutions. (L -glutamic acid) in LiCl solution has its transition at the same α value as in KCl solution. However, both the slopes of the pK_app versus α plots and the absolute values of ΔG°_hel are lower than in KCl solution. We interpret these results in terms of binding of Li⁺ to side chains as well as to the peptide bond.     

Enthalpy changes in the helix-coil transition of poly(gamma-benzyl L-glutamate)

The helix-coil transition temperature T_c of poly(γ-benzyl L -glutamate) in binary solvent mixtures of dichloroacetic acid and 1,4-dichlorobutane, 1-chlorooctane, or 1-chlorododecane have been measured. A treatment is presented with which the transition enthalpy can be calculated from the observed dependence of T_c on solvent composition. Results are compared with previously obtained calorimetric data. The underlying assumptions of the calculation are discussed.     

The helix-coil transitions of poly (dA)-poly (dT) and poly (dA-dT)-poly (dA-dT)

Helix–coil transition curves are calculated for poly (dA) poly(dT) and poly (dA-dT) poly (dA-dT) using the integral equation approach of Goel and Montroll.⁵ The transitions are described by the loop entropy model with the exponent of the loop entropy factor, k, remaining an arbitrary constant. The theoretical calculations are compared with experimental transition curves of the two polymers. Results indicate that the stacking energies for these two polymers differ by about 1 kcal/mole of base pairs. Also, a fit between theory and experiment was not possible for k > 1.70.     

Layer-by-layer Assembly of Poly(lactic-co-glycolic acid)-b-poly(l-lysine) Copolymer Micelles

Biocompatible, biodegradable polyionic micelles were used as a building component for layer-by-layer (LbL) assembly that can produce drug-loaded nanolayers. To prepare the polycationic micelles, poly(lactic-co-glycolic acid)-b-poly(l-lysine) [PLGA-b-P(Lys)] copolymers were synthesized. In an aqueous phase, PLGA-b-P(Lys) copolymers were self-assembled to form spherical micelles with the inner core of poly(lactic-co-glycolic acid) (PLGA) and the cationic outer shell of P(Lys). The micelles were characterized by zeta potential, dynamic light scattering, and nuclear magnetic resonance. PLGA-b-P(Lys) micelles showed the positive zeta potential values in a broad range of pH (3–11), indicating the high stability of the polyionic micelles with the outer shell of positive charges. Cationic polymeric micelles were coated on the surface via electrostatic interactions with the oppositely charged polyelectrolyte, poly(sodium 4-styrenesulfonate). Formation of multiple micelle layers was monitored using quartz crystal microbalance in situ, and the surface topology of the layers was characterized by atomic force microscopy ex situ, as the number of micelle layer was increased. The multiple micelle layers were stable, and the thickness of micelle layer grew as the number of LbL coating increased. The approach described in this work can be used for the development of the biocompatible, biodegradable, drug-loaded bioactive nanocoatings.     

NMR and optical activity observations on the helix-coil transition in poly(gamma-benzyl L-glutamate) solutions

The helix–coil transition of poly(γ-benzyl L -glutamate) was studied by comparing proton magnetic relaxation behavior with optical activity studies. The transition temperature as determined by magnetic relaxation was lower than that obtained by optical activity. The concentration dependence of the transition was also studied. The relationship of these experiments with previous NMR studies and also with calorimetric investigation of the transition is developed.     

Survival and maturation of cerebral neurons on poly(l-lysine) surfaces in the absence of serum

Several phenotypic properties of dissociated rat embryo cerebral cells cultivated in the absence of serum on poly(l-lysine) coated surfaces were investigated. A great enrichment of neuronal cells is attained after 3–4 weeks in culture as a result of cessation of nonneuronal cell proliferation. The monolayer pattern of cell-cell reassociation and neuritic sprouting is promoted by poly(l-lysine) and it is independent of serum presence or the existence of a layer of nonneuronal cells. Synaptic profiles and typical regions of neuropil are prominent. The neuronal plasma membrane is often characterized by irregular foldings at points of cell-cell contacts. A few biochemical parameters investigated lend support to the suggestion that the cerebral neurons acquire a certain state of functional maturation. It is proposed that substances secreted by the cells or released as a result of cell death may be conspicuous components for long-term survival of the cerebral neurons.     

Methodologies for monitoring nanoparticle formation by self-assembly of DNA with poly(l-lysine)

DNA self-assembly with polycations produces nanoparticles suitable for gene delivery, although there is no standard methodology to measure particle formation and stability. Here we have compared three commonly used assays, namely, light scattering, inhibition of ethidium bromide fluorescence, and modified electrophoretic mobility of DNA. Analysis by light scattering and loss of ethidium bromide fluorescence both showed poly(l-lysine) (pLL)/DNA nanoparticles form over the lysine/phosphate ratio range 0.6-1.0, although retardation of DNA electrophoretic mobility commenced at lower lysine/phosphate ratios. This probably indicates that the first two assays monitor DNA collapse into particles, while the electrophoresis assay measures neutralization of the charge on DNA. Gel analysis of the complexes showed disproportionation during nanoparticle formation, probably reflecting cooperative binding of the polycation. The assays were used to examine stability of complexes to dilution in water and physiological salts. Whereas all pLL/DNA nanoparticles were stable to dilution in water, the presence of physiological salts provoked selective disruption of complexes based on low-molecular-weight pLL. Polyelectrolyte complexes for targeted application in vivo should therefore be based on high-molecular-weight polycations, or should be stabilized to prevent their dissociation under physiological salt conditions.     

Laser Raman spectroscopic studies of poly(r-cytidylic acid) and poly(r-adenylic acid) complexes with poly(L-lysine) and poly(L-arginine)

Complexes of polyribocytidylic acid and polyriboadenylic acid with poly(L -lysine) and poly(L -arginine) were studied by Raman spectroscopy. The backbones of both polynucleotides are distorted by poly(L -arginine). On the other hand, poly(L -lysine) could distort the backbone of polyriboadenylic acid but not that of polyribocytidylic acid. In general, poly(L -arginine) can increase the order of the base stacking, while poly(L -lysine) causes disordering in the base stacking.     

Counterion binding in partially neutralized poly(D-glutamic acid) and poly(DL-glutamic acid)

Sodium counterion association with partially neutralized poly(D -glutamic acid) or poly(DL -glutamic acid) was measured by use of Wall's transference method with radioactive sodium. In the region where both polyacids are in completely random coil form, fractions of association were considerably less than that with poly(acrylic acid) in the same region of degree of neutralization. Even in the region where poly (D -glutamic acid) is in the helical form, the fraction of association was less than that with poly(acrylic acid) in the same region. No pronounced characteristics attributable to counterion association corresponding to the helix–coil transition could be found. The association phenomena were discussed on the basis of a rodlike model of polyelectrolyte.     

Kinetic studies on the helix-coil transition of fluorescent labeled poly(-L-lysine) by the temperature-jump technique

Fluorescent dansyl labels were covalently attached to poly (L-lysine) (poly(Lys)) with a degree of polymerization of 300 to 600. The degree of labeling was 0.01 to 0.085 (mol label to mol amino acid residues). From the decay of the anisotropy of fluorescence it was concluded that the labels were highly mobile both in the coiled and helical state. A decrease of fluorescence intensity accompanied the helix-coil transition. Identical pH induced transition curves were measured by circular dichroism and fluorescence. The midpoint of the transition was at pH 10.2. The kinetics of the transition were studied by temperature-jump relaxation using fluorescence detection. A single relaxation phase was observed. The relaxation time tau exhibited a distorted bell shaped dependence on the degree of helicity f with a maximum value tau(max) = 15 micros at f = 0.3 and 20 degrees C. It was independent of polymer concentration and of the degree of labeling. A rate constant of helix propagation kF = 10(7) s(-1) was calculated from tau(max) and published values of the nucleation parameter sigma. The activation energy was 16 kJ mol . The observed rate constant is comparable to that of poly(L-glutamic acid) but two orders of magnitude smaller than that found for polyamino acids with nonionizable side chains.     

Solid state microcellular foamed poly(lactic acid): morphology and property characterization

Poly(lactic acid) or PLA is a plant-based biodegrable plastic which exhibits many properties that are equivalent to or better than many petroleum-based plastics. However, there have been few commercial applications due to its lower impact resistance and higher cost than synthetic plastics. In this paper, the concept of creating microcellular foamed structures in PLA as a means to improve its shortcomings is presented. The effect of the foaming conditions (temperature and time) on the void fraction, volume expansion ratio, impact strength and tensile properties of foamed PLA is discussed. Each step of microcellular processing is addressed including: the manufacture of PLA film; the saturation of the samples with gas; the microcellular foaming of PLA; the void fraction determination, volume expansion ratio calculation, impact and tensile property characterization of foamed samples. The microcellular morphologies developed in PLA samples were a strong function of the foaming conditions. Due to the presence of foamed microcells, a twofold expansion ratio and significant improvements in the impact resistance (twofold increase over unfoamed PLA), strain at break (up to twofold increase over unfoamed PLA) and toughness (up to fourfold increase over unfoamed PLA) were achieved in PLA.     

An IR-investigation of the helix-coil conversion of poly U

In the presence of two or polyvalent cations, e. g., Mg²⁺, protonated ethylenediamine, protonated spermidine, and protonated spermine, poly U forms an ordered structure in aqueous solution. At elevated temperatures, the secondary structure is lost by a cooperative process. The melting temperature T_m rises with the size of the cations. Using IR-spectroscopy, we could show that the secondary structure of poly U is stabilized by hydrogen bonds. Both carbonyl groups of the uracil are involved in the hydrogen bonding. This experimental result enables us to choose the right arrangement for UU base pairing.     

Solution conformation of poly(L-lysyl-L-glutamic acid) and poly(L-lysyl-L-glutamine)

Solution conformation of poly(L-lysyl-L-glutamic acid) (PLGU) and poly(L-lysyl-L-glutamine) (PLGN) was studied in water as a function of pH, added salt, detergents, methanol and trifluoroethanol (TFE). Both the polypeptides exhibit no ordered conformation in the pH range 1.5-12.5; salts and detergents did not have any marked effect. Replacement of side chain carboxyl by an amide group did not help in inducing PLGN to adopt a helical conformation even at pH as high as 12.0, unlike poly(L-lysine). The helicogenic solvents, methanol and TFE, induce formation of weak helices in PLGU as well as in PLGN. It is not unlikely that H-bonding between the side chains leads to stabilizing an unordered conformations.     

Influence of poly(l-lysine) on the structure of dipalmitoylphosphatidylglycerol/water dispersions studied by X-ray scattering

The interaction between the negatively charged phospholipid DPPG and positively charged poly(l-lysine) (PLL) of different lengths was studied by X-ray scattering in the SAXS and WAXS region. As a reference pure DPPG (Na salt) was investigated over a wide temperature range (−30 to 70°C). The phase behavior of DPPG in aqueous and in buffer/salt dispersions showed a metastable subgel phase at low temperatures and a recrystallization upon heating before reaching the liquid–crystalline phase. The presence of additional salt stabilizes the bilayer structure and decreases the recrystallization temperature. Large changes in the SAXS region are not connected with changes in chain packing. In DPPG/PLL samples, the PLL is inserted between adjacent headgroup layers and liberates counterions which give rise to a freezing point depression. In the complex with DPPG PLL form an α-helical secondary structure at pH 7 and temperatures below the gel to liquid–crystalline phase transition. This prevents DPPG from recrystallization and strongly increases the stacking order. The lamellar repeat distance is decreased and fixed by the helix conformation of PLL in the gel phase. PLL with n = 14 is too short to form helices and is squeezed out reversibly from the interbilayer space upon cooling by freezing of trapped water. In dispersions with longer PLLs (n > 400) at −20°C a 1D crystallization of PLL α-helices in the aqueous layer between the headgroups takes place. A structural model is presented for the lateral periodic complex, which is similar to the known cationic lipid/DNA complex. Dedicated to Prof. K. Arnold on the occasion of his 65th birthday.     

Effect of redox-responsive DTSSP crosslinking on poly(l-lysine)-grafted-poly(ethylene glycol) nanoparticles for delivery of proteins

Therapeutic proteins are utilized in a variety of clinical applications, but side effects and rapid in vivo clearance still present hurdles. An approach that addresses both drawbacks is protein encapsulation within in a polymeric nanoparticle, which is effective but introduces the additional challenge of destabilizing the nanoparticle shell in clinically relevant locations. This study examined the effects of crosslinking self-assembled poly(l -lysine)-grafted-poly(ethylene glycol) nanoparticles with redox-responsive 3,3′-dithiobis(sulfosuccinimidyl propionate) (DTSSP) to achieve nanoparticle destabilization in a reductive environment. The polymer-protein nanoparticles (DTSSP NPs) were formed through electrostatic self-assembly and crosslinked with DTSSP, which contains a glutathione-reducible disulfide. As glutathione is upregulated in various cancers, DTSSP NPs could display destabilization within cancer cells. A library of DTSSP NPs was formed with varying copolymer to protein (C:P) and crosslinker to protein (X:P) mass ratios and characterized by size and encapsulation efficiency. DTSSP NPs with a 7:1 C:P ratio and 2:1 X:P ratio were further characterized by stability in the presence proteases and reducing agents. DTSSP NPs fully encapsulated the model protein and displayed 81% protein release when incubated with 5 mM dithiothreitol for 12 hr. This study contributes to understanding stimulus-responsive crosslinking of polymeric nanoparticles and could be foundational to clinical administration of therapeutic proteins.