Sulfonated polystyrenes: pH and Mg2+-insensitive amphiphilic copolymers for detergent-free membrane protein isolation.

Amphiphilic polymers are increasingly applied in the detergent-free isolation and functional studies of membrane proteins. However, the carboxylate group present in the structure of many popular variants, such as styrene-maleic acid (SMA) copolymers, brings limitations in terms of polymer sensitivity to precipitation at acidic pH or in the presence of divalent metal cations. Herein, we addressed this problem by replacing carboxylate with the more acidic sulfonate groups. To this end, we synthesized a library of amphiphilic poly[styrene-co-(sodium 4-styrene sulfonate)] copolymers (termed SSS), differing in their molecular weight and overall polarity. Using model cell membranes (Jurkat), we identified two copolymer compositions (SSS-L30 and SSS-L36) that solubilized membranes to an extent similar to SMA. Interestingly, the density gradient ultracentrifugation/SDS-PAGE/Western blotting analysis of cell lysates revealed a distribution of studied membrane proteins in the gradient fractions that was different than for SMA-solubilized membranes. Importantly, unlike SMA, the SSS copolymers remained soluble at low pH and in the presence of Mg2+ ions. Additionally, the solubilization of DMPC liposomes by the lead materials was studied by turbidimetry, DLS, SEC, and high-resolution NMR, revealing, for SSS-L36, the formation of stable particles (nanodiscs), facilitated by the direct hydrophobic interaction of the copolymer phenyls with lipid acyl chains.

Tailoring Butyl Methacrylate/Methacrylic Acid Copolymers for the Solubilization of Membrane Proteins: The Influence of Composition and Molecular Weight.

Low-molecular weight (MW) amphiphilic copolymers have been recently introduced as a powerful tool for the detergent-free isolation of cell membrane proteins. Herein, a screening approach is used to identify a new copolymer type for this application. Via a two-step ATRP/acidolysis procedure, a 3 × 3 matrix of well-defined poly[(butyl methacrylate)-co-(methacrylic acid)] copolymers (denoted BMAA) differing in their MW and ratio of hydrophobic (BMA) and hydrophilic (MAA) units is prepared. Subsequently, using the biologically relevant model (T-cell line Jurkat), two compositions of BMAA copolymers are identified that solubilize cell membranes to an extent comparable to the industry standard, styrene-maleic acid copolymer (SMA), while avoiding the potentially problematic phenyl groups. Surprisingly, while only the lowest-MW variant of the BMA/MAA 2:1 composition is effective, all the copolymers of the BMA/MAA 1:1 composition are found to solubilize the model membranes, including the high-MW variant (MW of 14 000). Importantly, the density gradient ultracentrifugation/sodium dodecyl sulfate-polyacrylamide gel electrophoresis/Western blotting experiments reveal that the BMA/MAA 1:1 copolymers disintegrate the Jurkat membranes differently than SMA, as demonstrated by the different distribution patterns of two tested membrane protein markers. This makes the BMAA copolymers a useful tool for studies on membrane microdomains differing in their composition and resistance to membrane-disintegrating polymers.

Fast and efficient single step liquid chromatography separation of parent homopolymers from block copolymers.

The modified layout of the barrier method called liquid chromatography under limiting conditions of enthalpic interactions is presented. It enables automated quantitative separation of blends of synthetic polymers, for example the single step discrimination of both parent homopolymers from the block copolymers. Moreover, this method enables the estimation of molar mass and molar mass distribution of the block copolymer precursor. Adjacent large sequences of mobile phase of different composition are applied as barriers. They are created by a computer controlled pair of pumping systems in the form of longitudinal profiles along the column. The home synthesized block copolymers polystyrene-block-poly(2-vinylpyridine) served as model examples of the method application. The adsorption retention mechanism was exploited using mesoporous bare silica gel column packing. Series of block copolymers of similar composition can be quickly handled with the method to optimize their synthesis.

Nano-modified epoxy: the effect of GO-based complex structures on mechanical performance.

The application of nanofillers (NFs) in multicomponent polymer systems is accompanied by important structure-directing effects that are more marked in partially miscible systems, such as polymer-modified epoxy. This study deals with rubber-modified epoxy using different combinations of GO and amine-terminated butadiene-acrylonitrile copolymer (ATBN), including in situ and pre-made grafting. Moreover, GO grafted via planar epoxy groups or solely edge-localized carboxyls was used. It is shown that the grafted ATBN chains promote the assembly of GO-g-ATBN into nacre-mimicking lamellar structures instead of usual exfoliation in thermoplastics. This complex structure of elastically embedded GO leads to the best mechanical performance. It is obvious that a small concentration of the grafted polymer exceeds the contribution of a higher concentration of separately added ATBN. The results highlight the important effect of the degree of grafted chains and geometry of the internal structure of the self-assembled arrays and their effect on the mechanical performance.

MALDI-ToF mass spectrometry detection of intramolecular composition gradient in copolymers.

Since their addition to the polymer-architecture portfolio, gradient copolymers have attracted significant attention. Up to now, however, the existence of the intramolecular composition gradient must have been ascertained by sampling during living copolymerization because a reliable method for the detection of the composition gradient in the finalized copolymer had not been established yet. Here we show that MALDI-ToF mass spectrometry not only identifies imperfect, i.e. prematurely terminated copolymers but these copolymers can be used as "time capsules" which provide information on composition evolution and the intramolecular composition gradient.

Formation of core/corona nanoparticles with interpolyelectrolyte complex cores in aqueous solution: insight into chain dynamics in the complex from fluorescence quenching.

Formation of interpolyelectrolyte complexes (IPECs) of poly(methacrylic acid) (PMAA) bearing a fluorescent label (umbelliferone) at the chain end and poly[3,5-bis(trimethyl ammoniummethyl)-4-hydroxystyrene iodide]-block-poly(ethylene oxide) (QNPHOS-PEO) acting as a fluorescence quencher, was followed using a combination of scattering, calorimetry, microscopy and fluorescence spectroscopy techniques. While scattering and microscopy measurements indicated formation of spherical core/corona nanoparticles with the core of the QNPHOS/PMAA complex and the PEO corona, fluorescence measurements showed that both static and dynamic quenching efficiency were increased in the nanoparticle stability region. As the dynamic quenching rate constant remained unchanged, the quenching enhancement was caused by the increase in the local concentration of QNPHOS segments in the microenvironment of the label. This finding implies that the local dynamics of PMAA end chains affecting the interaction of the label with QNPHOS segments was independent of both PMAA and QNPHOS chain conformations.

Local pH and Effective pK of a Polyelectrolyte Chain: Two Names for One Quantity?

In recent experiments, the "local pH" near polyelectrolyte chains was determined from the shift in the effective acidity constant of fluorescent pH indicators attached to the macromolecules. This indirect determination raises the question if the analyzed quantity was indeed the "local pH" and what this term actually means. In this study, we combined experiments and simulations to demonstrate that the shift in ionization constant is slightly lower than the difference between the pH and the "local pH". This offset is caused by correlations between fluctuations in chain conformation, small-ion distribution, and fluorophore ionization.

Decomposition of size-exclusion chromatography elution curves of complex branched polymers.

A new method for the decomposition of non-baseline-resolved multimodal elution curves of SEC with the concentration, light scattering and viscosity detection is presented. The method makes possible the characterization of the polymer-sample components, represented by the peaks forming multimodal elution curves, individually and reduces also the error in the calculation of molecular-weight averages. The procedure is demonstrated on narrow molecular-weight distribution polystyrene standards and their mixture as well as on a grafted polymer sample.

Self-assembly of poly(4-methylstyrene)-g-poly(methacrylic acid) graft copolymer in selective solvents for grafts: scattering and molecular dynamics simulation study.

Poly(4-methylstyrene)-g-poly(methacrylic acid) (P4MS-g-PMAA) graft copolymer was synthesized by the grafting-onto method from poly(4-methylstyrene), selectively brominated on methyl groups, and "living" poly(tert-butyl methacrylate). The average degree of polymerization of the backbone and the grafts and the average number of grafts per backbone were 251, 21, and 25, respectively. The self-assembly of P4MS-g-PMAA was studied in methanol and aqueous buffers (selective solvents for grafts). Micelles of P4MS-g-PMAA in methanol were studied by a combination of static and dynamic light scattering, TEM and SAXS. It was found that their spherical core/shell morphology resembles that of diblock copolymer micelles but they have a very low aggregation number (approximately 3) and a high cmc (approximately 0.8 mg/mL). The spherical core-shell structure revealed by SAXS was confirmed by the molecular dynamics study emulating an associate of comblike copolymers with structural parameters close to those of the experimentally studied system. Because P4MS-g-PMAA was not directly soluble in water, its aqueous solutions had to be prepared by dialysis of the methanolic solutions. In aqueous solutions, unlike in methanol, small P4MS-g-PMAA micelles (approximately 20 nm in diameter) form large secondary aggregates (approximately 100-400 nm).