A theory for the displacement of proteins and viruses with polyethylene glycol.

The displacement action of polyethylene glycol of different molecular weights may be linked to the ability of the polymers to form coiled particles in solution. From conclusions drawn from their sedimentating properties in centrifugal fields the polyethylene glycols of low molecular weights, as expected, are less randomly coiled than those of higher molecular weight. It is suggested that protein molecules have the ability to diffuse into the coils of the polyethylene glycol from which they are excluded when the random coiling increases with increasing polymer concentration. From considerations based on the interaction of the polymer filament with the displaced particle the distribution of the substance between the coils and the intermolecular spaces may be predicted semi-quantitatively.     

A Theory for the Displacement of Proteins and Viruses with Polyethylene Glycol

The displacement action of polyethylene glycol of different molecular weights may be linked to the ability of the polymers to form coiled particles in solution. From conclusions drawn from their sedimentating properties in centrifugal fields the polyethylene glycols of low molecular weights, as expected, are les randomly coiled than those of higher molecular weight. It is suggested that protein molecules have the ability to diffuse into the coils of the polyethylene glycol from which they are excluded when the random coiling increases with increasing polymer concentration. From considerations based on the interaction of the polymer filament with the displaced particle the distribution of the substance between the coils and the intermolecular spaces may be predicted semi-quantitatively.     

Prediction of the stability of coiled coils using molecular dynamics simulations

We have performed 40–80 ns-long molecular dynamics (MD) simulations of the GCN4 leucine zipper and synthetic coiled coils using the GROMOS96 (43a2) and OPLS-AA force fields, with the aim of predicting coiled coil stability. Starting with an initial configuration of two peptides placed in an ideal coiled coil configuration, we find that changing the amino acid sequence modestly or decreasing peptide length can lead to a decrease in the final α-helicity of coiled coils, although for peptides as long or longer than 16 residues, the values of helicity do not decrease to the low values seen in the experimental results of Lumb et al. (Biochemistry. 1994, 33, 7361–7367) or of Su et al. (Biochemistry. 1994, 33, 15501–15510), presumably because the simulations are not long enough. We find, however, that helicity correlates positively with the number of close hydrophobic interactions between the two peptides, showing that stable coiled coils in the simulations are tightly packed. The minimum interhelical distances are 0.50–0.66 nm for charged groups, indicating that favorable charge interactions are also important for the stability of the coiled coil.     

17. Purification and Aggregation of Influenza Virus by Precipitation with Polyethylene Glycol

ABSTRACT

Influenza virus may be purified and rendered free of extraneous proteins by precipitation and aggregation with polyethylene glycol at polymer concentrations of 1 to 4%. The precipitated virus is superior antigenically to the virus in monomeric and in the ether dissociated forms. When the virus is precipitated at polyethylene glycol concentrations of 5% and higher the virus is not aggregated and is associated with extraneous protein which co-precipitates with the infectious agent.     

Cryoprotection of purified rat kidney transamidinase by polyethylene glycol.

Polyethylene glycol is a water-soluble polymer which is widely used in the pharmaceutical, cosmetic, and chemical industries. In this study, it is shown that polyethylene glycol is an effective cryoprotectant of rat kidney transamidinase purified from both the mitochondria and cytosol. Much of the activity is lost when the purified enzyme is frozen and thawed in sodium-potassium phosphate buffer in the absence of cryoprotectants. Polyethylene glycols with molecular weights of 4000 to 10,000 were effective cryoprotectants. However, polyethylene glycols with a molecular weight of 1000 or lower inhibited the purified enzyme. A concentration of only 0.01% polyethylene glycol 4000, 8000, or 10,000 was required for complete cryoprotection. In addition to polyethylene glycol, 0.5 mM ethylenediaminetetraacetic acid was required in the phosphate buffer for complete cryoprotection. The stabilization of purified transamidinase by polyethylene glycol will facilitate characterization experiments designed to compare the properties of the mitochondrial and cytosolic isozymes.     

Degradation of ethylene glycol and polyethylene glycols by methanogenic consortia.

Methanogenic enrichments capable of degrading polyethylene glycol and ethylene glycol were obtained from sewage sludge. Ethanol, acetate, methane, and (in the case of polyethylene glycols) ethylene glycol were detected as products. The sequence of product formation suggested that the ethylene oxide unit [HO-(CH2-CH2-O-)xH] was dismutated to acetate and ethanol; ethanol was subsequently oxidized to acetate by a syntrophic association that produced methane. The rates of degradation for ethylene, diethylene, and polyethylene glycol with molecular weights of 400, 1,000, and 20,000, respectively, were inversely related to the number of ethylene oxide monomers per molecule and ranged from 0.84 to 0.13 mM ethylene oxide units degraded per h. The enrichments were shown to best metabolize glycols close to the molecular weight of the substrate on which they were enriched. The anaerobic degradation of polyethylene glycol (molecular weight, 20,000) may be important in the light of the general resistance of polyethylene glycols to aerobic degradation.     

The effect of molecular size, concentration in nutrient solution, and exposure time on the amount and distribution of polyethylene glycol in pepper plants

Pepper plants Capsicum annuum L. var. California Wonder were grown in nutrient solutions of either −3.0 or −5.0 bars osmotic potential, using polyethylene glycol with molecular weights of 400, 600, 1000, 1540, or 4000 as osmotica. Polyethylene glycol with molecular weights of 1000 or 1540 proved most satisfactory as osmotica to decrease the water potential of nutrient solutions.     

Allowing for polymer polydispersity--an essential condition for determination of aqueous pore diameters in cell walls and membranes using polymers

A method of allowing for polydispersion of polyethylene glycol (PEG) preparations was developed for the use of these preparations for the osmometrical evaluation of pore diameters with aqueous pores of Chara corallina cell walls as an example. The mass share of polyethylene glycol preparation fractions gamma p penetrating through the pores was determined using cellular "shadows", fragments of internodal cell walls tied up at the ends and filled with a 25% solution of nonpenetrating PEG 6000. When immersed into water, such "shadow" acquired a turgor (hydrostatic) pressure close to the cellular pressure and persistent over long time. The determination of gamma p for polyethylene glycols with different average molecular weights Mw was performed from the degree of pressure restoration after water was replaced by a 5-10% polymer solution. The kinetics of pressure changes was recorded using a mechanotronic dynamometer, which measures, in the quasi-isometric mode, the force necessary for partial compression of the "shadow" in its small fragment. By utilizing the dependence of the overall share of fractions with molecular weights Mi < Mk on Mk (data of [1]), we found that gamma p, for these polyethylene glycols corresponds to the threshold value of Mk = 800-1100 D (hydrodynamic radius of molecules rh = 0.85-1.05 nm). Thus, the effective diameter of the pores in the cell wall of Chara does not exceed 2.1 nm. It was shown that the smoothness of the sigmoid shape of the dependence of ionic channel conductivity on the Mw value of the polymer in the media is largely due to the polydispersion of polymer preparations, particularly, to the reduction in the share of fractions penetrating the channels as Mw is increased. The method normally used to estimate pore diameters in ionic channels which ignores the dispersion of polymer preparations, results in overestimated values.     

Polyethylene glycol in aqueous solution: Solvent perturbation and gel filtration studies

As part of an investigation of the mechanism of precipitation of proteins by synthetic polymers, we measured the ability of oligomers and polymers of ethylene glycol to (a) alter the solubility of amino acids, (b) perturb the absorption spectra of aromatic amino acids, and (c) enhance the fluorescence of 1,8-anilinonaphthalene sulfonate (ANS). All of these effects increased with increasing degree of polymerization, up to a molecular weight of about 400. This trend can be partially attributed to the diminishing influence of the terminal OH groups. However, small analogs such as dioxane and dimethoxyethane (lacking OH groups) were much less effective than the polymers, suggesting a cooperative effect between neighboring ethoxide residues in the polymer. In contrast, the effectiveness of polyethylene glycol (PEG) as a protein-precipitating agent continued to increase with increasing size; polymers of molecular weight 4000–6000 were substantially more effective than their 10-fold smaller homologues. On exclusion chromatographic columns, the synthetic polymers eluted much earlier than did proteins of comparable molecular weight. This discrepancy diminished in 6 m guanidine where the proteins eluted much earlier, reflecting their conversion to random coils. In contrast, the elution of PEG was only slightly affected, suggesting a random coil configuration even in the absence of denaturant. PEG-20M, a mixture prepared by coupling 2 mol of PEG-6000 with an epoxide, was resolved into two components by exclusion chromatography on Sephadex G-100. The fast-eluting component (the coupled product) was unusually effective in enhancing ANS fluorescence and was shown to bind the dye with K_a = 5.1 × 10³, M^?1 at 25 °C.     

Identification of two distinct heparin cofactors in human plasma: Separation and partial purification

Two distinct components having potent heparin cofactor activity have been separated from human blood plasma and partially purified by means of precipitation with polyethylene glycol, adsorption on Al(OH)₃ gel and ion exchange chromatography on DEAE-cellulose and hydroxyapatite. The first component, cofactor A, is distinguished from the second, cofactor B, by its very low activity in the progressive antithrombin assay. Ion exchange rechromatography experiments indicate that cofactors A and B are not interconvertible, and that neither is an artifact of the polyethylene glycol precipitation step. Molecular weights, estimated by gel filtration were found to be 105,000 and 87,000 daltons for cofactors A and B respectively. Except for the molecular weight, the bulk of evidence suggests that cofactor B and the antithrombin III purified by Abildgaard (3) are identical.     

Exploring alternate states and oligomerization preferences of coiled‐coils by de novo structure modeling

Homomeric coiled‐coils can self‐assemble into a wide range of structural states with different helix topologies and oligomeric states. In this study, we have combined de novo structure modeling with stability calculations to simultaneously predict structure and oligomeric states of homomeric coiled‐coils. For dimers an asymmetric modeling protocol was developed. Modeling without symmetry constraints showed that backbone asymmetry is important for the formation of parallel dimeric coiled‐coils. Collectively, our results demonstrate that high‐resolution structure of coiled‐coils, as well as parallel and antiparallel orientations of dimers and tetramers, can be accurately predicted from sequence. De novo modeling was also used to generate models of competing oligomeric states, which were used to compare stabilities and thus predict the native stoichiometry from sequence. In a benchmark set of 33 coiled‐coil sequences, forming dimers to pentamers, up to 70% of the oligomeric states could be correctly predicted. The calculations demonstrated that the free energy of helix folding could be an important factor for determining stability and oligomeric state of homomeric coiled‐coils. The computational methods developed here should be broadly applicable to studies of sequence‐structure relationships in coiled‐coils and the design of higher order assemblies with improved oligomerization specificity. Proteins 2015; 83:235–247. © 2014 Wiley Periodicals, Inc.     

Computational assessment of folding energy landscapes in heterodimeric coiled coils

The coiled coil structural motif consists of alpha helices supercoiling around each other to form staggered knobs‐into‐holes packing. Such structures are deceptively simple, especially as they often can be described with parametric equations, but are known to exist in various conformations. Even the simplest systems, consisting of 2 monomers, can assemble into a wide range of states. They can form canonical as well as noncanonical coiled coils, be parallel or antiparallel, where helices associate with different degrees of shift, tilt, and rotation. Here, we investigate the energy landscape of heterodimeric coiled coils by carrying out de novo folding simulations starting from amino acid sequence. We folded a diverse set of 22 heterodimers and demonstrate that the approach is capable of identifying the atomic details in the experimental structure in the majority of cases. Our methodology also enables exploration of alternative states that can be accessible in solution beyond the experimentally determined structure. For many systems, we observe folding energy landscapes with multiple energy minima and several isoenergetic states. By comparing coiled coils from single domains and those extracted from larger proteins, we find that standalone coiled coils have deeper energy wells at the experimentally determined conformation. By folding the competing homodimeric states in addition to the heterodimers, we observe that the structural specificity towards the heteromeric state is often small. Taken together, our results demonstrate that de novo folding simulations can be a powerful tool to characterize structural specificity of coiled coils when coupled to assessment of energy landscapes.     

Branched Polyethylene Glycol (Bpeg) Conjugated Antisense Oligonucleotides

Abstract

The synthesis of new oligonucleotide conjugates bearing an high molecular weight, branched polyethylene glycol (bPEG) chain is reported.     

Fusarium solani pisi recombinant cutinase partitioning in peg/potassium phosphate aqueous two-phase systems

Summary An aqueous two-phase system of polyethylene glycol (PEG) and potassium phosphate was developed for extraction of a cutinase from cell debris of a recombinant Escherichia coli strain. Basic studies to identify the primary factors which affect cutinase partition, namely the influence of polymer molecular weight, polymer concentration and pH were carried out using a purified preparation of the cutinase. The enzyme partition coefficient was enhanced with decreasing PEG molecular weight, increasing tie-line length and pH.     

Hierarchical Cascades of Instability Govern the Mechanics of Coiled Coils: Helix Unfolding Precedes Coil Unzipping

Coiled coils are a fundamental emergent motif in proteins found in structural biomaterials, consisting of α-helical secondary structures wrapped in a supercoil. A fundamental question regarding the thermal and mechanical stability of coiled coils in extreme environments is the sequence of events leading to the disassembly of individual oligomers from the universal coiled-coil motifs. To shed light on this phenomenon, here we report atomistic simulations of a trimeric coiled coil in an explicit water solvent and investigate the mechanisms underlying helix unfolding and coil unzipping in the assembly. We employ advanced sampling techniques involving steered molecular dynamics and metadynamics simulations to obtain the free-energy landscapes of single-strand unfolding and unzipping in a three-stranded assembly. Our comparative analysis of the free-energy landscapes of instability pathways shows that coil unzipping is a sequential process involving multiple intermediates. At each intermediate state, one heptad repeat of the coiled coil first unfolds and then unzips due to the loss of contacts with the hydrophobic core. This observation suggests that helix unfolding facilitates the initiation of coiled-coil disassembly, which is confirmed by our 2D metadynamics simulations showing that unzipping of one strand requires less energy in the unfolded state compared with the folded state. Our results explain recent experimental findings and lay the groundwork for studying the hierarchical molecular mechanisms that underpin the thermomechanical stability/instability of coiled coils and similar protein assemblies.     

Hierarchical Cascades of Instability Govern the Mechanics of Coiled Coils: Helix Unfolding Precedes Coil Unzipping

Coiled coils are a fundamental emergent motif in proteins found in structural biomaterials, consisting of α-helical secondary structures wrapped in a supercoil. A fundamental question regarding the thermal and mechanical stability of coiled coils in extreme environments is the sequence of events leading to the disassembly of individual oligomers from the universal coiled-coil motifs. To shed light on this phenomenon, here we report atomistic simulations of a trimeric coiled coil in an explicit water solvent and investigate the mechanisms underlying helix unfolding and coil unzipping in the assembly. We employ advanced sampling techniques involving steered molecular dynamics and metadynamics simulations to obtain the free-energy landscapes of single-strand unfolding and unzipping in a three-stranded assembly. Our comparative analysis of the free-energy landscapes of instability pathways shows that coil unzipping is a sequential process involving multiple intermediates. At each intermediate state, one heptad repeat of the coiled coil first unfolds and then unzips due to the loss of contacts with the hydrophobic core. This observation suggests that helix unfolding facilitates the initiation of coiled-coil disassembly, which is confirmed by our 2D metadynamics simulations showing that unzipping of one strand requires less energy in the unfolded state compared with the folded state. Our results explain recent experimental findings and lay the groundwork for studying the hierarchical molecular mechanisms that underpin the thermomechanical stability/instability of coiled coils and similar protein assemblies.     

The Evolution and Structure Prediction of Coiled Coils across All Genomes

Coiled coils are α-helical interactions found in many natural proteins. Various sequence-based coiled-coil predictors are available, but key issues remain: oligomeric state and protein-protein interface prediction and extension to all genomes. We present SpiriCoil (http://supfam.org/SUPERFAMILY/spiricoil), which is based on a novel approach to the coiled-coil prediction problem for coiled coils that fall into known superfamilies: hundreds of hidden Markov models representing coiled-coil-containing domain families. Using whole domains gives the advantage that sequences flanking the coiled coils help. SpiriCoil performs at least as well as existing methods at detecting coiled coils and significantly advances the state of the art for oligomer state prediction. SpiriCoil has been run on over 16 million sequences, including all completely sequenced genomes (more than 1200), and a resulting Web interface supplies data downloads, alignments, scores, oligomeric state classifications, three-dimensional homology models and visualisation. This has allowed, for the first time, a genomewide analysis of coiled-coil evolution. We found that coiled coils have arisen independently de novo well over a hundred times, and these are observed in 16 different oligomeric states. Coiled coils in almost all oligomeric states were present in the last universal common ancestor of life. The vast majority of occasions that individual coiled coils have arisen de novo were before the last universal common ancestor of life; we do, however, observe scattered instances throughout subsequent evolutionary history, mostly in the formation of the eukaryote superkingdom. Coiled coils do not change their oligomeric state over evolution and did not evolve from the rearrangement of existing helices in proteins; coiled coils were forged in unison with the fold of the whole protein.     

Potentiation of mitogen-induced lymphocyte stimulation by polyethylene glycols

The effect of polyethylene glycols of different molecular weights on mitogen-induced lymphocyte stimulation was investigated. The stimulation obtained by neuraminidase/galactose oxidase treatment, and by addition of several plant lectin mitogens, was markedly enhanced in the presence of polyethylene glycols of molecular weights ranging from 6 000 to 40 000. Optimal potentiation of stimulation was found for polyethylene glycol concentrations up to about 5 %.