Polyelectrolyte Complex Coacervation across a Broad Range of Charge Densities.
Ontology highlight
ABSTRACT:
SUBMITTER: Neitzel AE
PROVIDER: S-EPMC8320234 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Proteins are an important class of biologics, but there are several recurring challenges to address when designing protein-based therapeutics. These challenges include: the propensity of proteins to aggregate during formulation, relatively low loading in traditional hydrophobic delivery vehicles, and inefficient cellular uptake. This last criterion is particularly challenging for anionic proteins as they cannot cross the anionic plasma membrane. Here we investigated the complex coacervation of anionic proteins with a block copolymer of opposite charge to form polyelectrolyte complex (PEC) micelles for use as a protein delivery vehicle. Using genetically modified variants of the model protein green fluorescent protein (GFP), we evaluated the role of protein charge and charge localization in the formation and stability of PEC micelles. A neutral-cationic block copolymer, poly(oligoethylene glycol methacrylate-block-quaternized 4-vinylpyridine), POEGMA79-b-qP4VP175, was prepared via RAFT polymerization for complexation and microphase separation with the panel of engineered anionic GFPs. We found that isotropically supercharged proteins formed micelles at higher ionic strength relative to protein variants with charge localized to a polypeptide tag. We then studied GFP delivery by PEC micelles and found that they effectively delivered the protein cargo to mammalian cells. However, cellular delivery varied as a function of protein charge and charge distribution and we found an inverse relationship between the PEC micelle critical salt concentration and delivery efficiency. This model system has highlighted the potential of polyelectrolyte complexes to deliver anionic proteins intracellularly. Using this model system, we have identified requirements for the formation of PEC micelles that are stable at physiological ionic strength and that smaller protein-polyelectrolyte complexes effectively deliver proteins to Jurkat cells.
| S-EPMC9749388 | biostudies-literature
Project description:In this work, polyelectrolyte mixing ratio is studied as a tuning parameter to control the charge, and thus the separation properties of polyelectrolyte complex (PEC) membranes prepared via Aqueous Phase Separation (APS). In this approach, various ratios of poly(sodium 4-styrenesulfonate) (PSS) and poly(diallyldimethylammonium chloride) (PDADMAC) are mixed at high salinity and the PEC-based membranes are then precipitated using low salinity coagulation baths. The monomeric ratio of PSS to PDADMAC is varied from 1.0 : 0.8 through to 1.0 : 1.2. Obtained membranes have an asymmetric structure and function as nanofiltration membranes with on average 1 L m-2 h-1 bar-1 pure water permeance and <400 Da molecular weight cut-off (MWCO); except for the 1.0 : 1.2 membrane, where the water permeance was much higher (>20 L m-2 h-1 bar-1) with a similarly low MWCO. For the first time, we report the formation of both negatively and positively charged PSS-PDADMAC based APS membranes, as determined by both streaming potential and salt retention measurements. We hypothesize that the salt type used in the APS process plays a key role in the observed change in membrane charge. The point where the membrane charge transitions from negative to positive is found to be between the 1.0 : 0.9 and 1.0 : 1.0 PSS : PDADMAC ratios. The polyelectrolyte ratio not only affects membrane charge, but also their mechanical properties. The 1.0 : 0.9 and 1.0 : 1.0 membranes perform the best amongst the membranes prepared in this study since they have high salt retentions (up to 90% Na2SO4 and 75% MgCl2, respectively) and better mechanical stability. The higher permeance of the more charged, and thus more swollen, 1.0 : 0.8 and 1.0 : 1.2 membranes provide a relevant new direction for the development of APS-based PEC membranes.
| S-EPMC8549507 | biostudies-literature
Project description:A procedure to derive the electrostatic potential (ESP) for dynamic charge densities obtained from structure models or maximum-entropy densities is introduced. The ESP essentially is obtained by inverse Fourier transform of the dynamic structure factors of the total charge density corresponding to the independent atom model, the multipole model or maximum-entropy densities, employing dedicated software that will be part of the BayMEM software package. Our approach is also discussed with respect to the Ewald summation method. It is argued that a meaningful ESP can only be obtained if identical thermal smearing is applied to the nuclear (positive) and electronic (negative) parts of the dynamic charge densities. The method is applied to structure models of dl-serine at three different temperatures of 20, 100 and 298 K. The ESP at locations near the atomic nuclei exhibits a drastic reduction with increasing temperature, the largest difference between the ESP from the static charge density and the ESP of the dynamic charge density being at T = 20 K. These features demonstrate that zero-point vibrations are sufficient for changing the spiky nature of the ESP at the nuclei into finite values. On 0.5 e Å-3 isosurfaces of the electron densities (taken as the molecular surface relevant to intermolecular interactions), the dynamic ESP is surprisingly similar at all temperatures, while the static ESP of a single molecule has a slightly larger range and is shifted towards positive potential values.
| S-EPMC5713142 | biostudies-literature
Project description:In this study, the chitosan-induced coacervation of soy protein-isoflavone complexes in soymilk was investigated. Most of the soymilk proteins, including ?-conglycinin (7S), glycinin (11S), and isoflavones, were found to coacervate into the soymilk pellet fraction (SPF) following the addition of 0.5% chitosan. The total protein in the soymilk supernatant fraction (SSF) decreased from 18.1 ± 0.3 mg/mL to 1.6 ± 0.1 mg/mL, and the pH values decreased slightly, from 6.6 ± 0.0 to 6.0 ± 0.0. The results of SDS-PAGE revealed that the 7S ?', 7S ?, 7S ?, 11S A3, and 11S acidic subunits, as well as the 11S basic proteins in the SSF, decreased to 0.7 ± 0.5%, 0.2 ± 0.1%, 0.1 ± 0.0%, 0.2 ± 0.2%, 0.2 ± 0.2% and 0.3 ± 0.2%, respectively. We also found that isoflavones in the SSF, including daidzein, glycitein, and genistein, decreased to 9.6 ± 2.3%, 5.7 ± 0.9% and 5.9 ± 1.5%, respectively. HPLC analysis indicated that isoflavones mixed with soy proteins formed soy protein-isoflavone complexes and were precipitated into the SPF by 0.5% chitosan.
| S-EPMC6152765 | biostudies-literature
Project description:Widespread vaccine coverage for viral diseases could save the lives of millions of people each year. For viral vaccines to be effective, they must be transported and stored in a narrow temperature range of 2-8 °C. If temperatures are not maintained, the vaccine may lose its potency and would no longer be effective in fighting disease; this is called the cold storage problem. Finding a way to thermally stabilize a virus and end the need to transport and store vaccines at refrigeration temperatures will increase access to life-saving vaccines. We explore the use of polymer-rich complex coacervates to stabilize viruses. We have developed a method of encapsulating virus particles in liquid complex coacervates that relies on the electrostatic interaction of viruses with polypeptides. In particular, we tested the incorporation of two model viruses; a non-enveloped porcine parvovirus (PPV) and an enveloped bovine viral diarrhea virus (BVDV) into coacervates formed from poly(lysine) and poly(glutamate). We identified optimal conditions (i.e., the relative amount of the two polypeptides) for virus encapsulation, and trends in this composition matched differences in the isoelectric point of the two viruses. Furthermore, we were able to achieve a ?103-104-fold concentration of virus into the coacervate phase, such that the level of virus remaining in the bulk solution approached our limit of detection. Lastly, we demonstrated a significant enhancement of the stability of non-enveloped PPV during an accelerated aging study at 60 °C over the course of a week. Our results suggest the potential for using coacervation to aid in the purification and formulation of both enveloped and non-enveloped viruses, and that coacervate-based formulations could help limit the need for cold storage throughout the transportation and storage of vaccines based on non-enveloped viruses.
| S-EPMC7797030 | biostudies-literature
Project description:Complex coacervation refers to the formation of distinct liquid phases that arise when polyelectrolytes are mixed under appropriate polymer and salt concentrations. Molecular-level studies of coacervation have been limited. In this work, a coarse-grained model of the polymers and the corresponding counterions is proposed and used to simulate coacervation as a function of polymer length and overall salt concentration. Several sampling methods are used to determine the phase behavior of the underlying polymers. In particular, the results of simulations in different ensembles are shown to be consistent and to reproduce a number of phenomena observed in experiments, including the disruption of complexation by increasing ionic strength or by decreasing molecular weight. The coacervate concentrations determined from phase behavior calculations are then used to examine the rheology of the corresponding materials. By relying on long dynamic simulations, we are able to generate the dynamic response of the material in the form of dynamic moduli as a function of frequency, which are also found to compare favorably with experimental measurements.
| S-EPMC7722290 | biostudies-literature
Project description:Cytoplasmic inclusions containing aberrant proteolytic fragments of TDP-43 are associated with frontotemporal lobar degeneration (FTLD) and other related pathologies. In FTLD, TDP-43 is translocated into the cytoplasm and proteolytically cleaved to generate a prion-like domain (PrLD) containing C-terminal fragments (C25 and C35) that form toxic inclusions. Under stress, TDP-43 partitions into membraneless organelles called stress granules (SGs) by coacervating with RNA and other proteins. To study the factors that influence the dynamics between these cytoplasmic foci, we investigated the effects of cysteine-rich granulins (GRNs 1-7), which are the proteolytic products of progranulin, a protein implicated in FTLD, on TDP-43. We show that extracellular GRNs, typically generated during inflammation, internalize and colocalize with PrLD as puncta in the cytoplasm of neuroblastoma cells but show less likelihood of their presence in SGs. In addition, we show GRNs and PrLD coacervate to undergo liquid-liquid phase separation (LLPS) or form gel- or solid-like aggregates. Using charge patterning and conserved cysteines among the wild-type GRNs as guides, along with specifically engineered mutants, we discover that the negative charges on GRNs drive LLPS while the positive charges and the redox state of cysteines modulate these phase transitions. Furthermore, RNA and GRNs compete and expel one another from PrLD condensates, providing a basis for GRN's absence in SGs. Together, the results help uncover potential modulatory mechanisms by which extracellular GRNs, formed during chronic inflammatory conditions, could internalize and modulate cytoplasmic TDP-43 inclusions in proteinopathies.
| S-EPMC9247478 | biostudies-literature
Project description:A set of modular broad-host-range expression vectors with various affinity tags (six-His-tag, FLAG-tag, Strep-tag II, T7-tag) was created. The complete nucleotide sequences of the vectors are known, and these small vectors can be mobilized by conjugation. They are useful in the purification of proteins and protein complexes from gram-negative bacterial species. The plasmids were easily customized for Thiocapsa roseopersicina, Rhodobacter capsulatus, and Methylococcus capsulatus by inserting an appropriate promoter. These examples demonstrate the versatility and flexibility of the vectors. The constructs harbor the T7 promoter for easy overproduction of the desired protein in an appropriate Escherichia coli host. The vectors were useful in purifying different proteins from T. roseopersicina. The FLAG-tag-Strep-tag II combination was utilized for isolation of the HynL-HypC2 protein complex involved in hydrogenase maturation. These tools should be useful for protein purification and for studying protein-protein interactions in a range of bacterial species.
| S-EPMC348848 | biostudies-literature
Project description:The intrinsically disordered region (IDR) is a preserved signature of phytobacterial type III effectors (T3Es). The T3E IDR is thought to mediate unfolding during translocation into the host cell and to avoid host defense by sequence diversification. Here, we demonstrate a mechanism of host subversion via the T3E IDR. We report that the Xanthomonas campestris T3E XopR undergoes liquid-liquid phase separation (LLPS) via multivalent IDR-mediated interactions that hijack the Arabidopsis actin cytoskeleton. XopR is gradually translocated into host cells during infection and forms a macromolecular complex with actin-binding proteins at the cell cortex. By tuning the physical-chemical properties of XopR-complex coacervates, XopR progressively manipulates multiple steps of actin assembly, including formin-mediated nucleation, crosslinking of F-actin, and actin depolymerization, which occurs through competition for actin-depolymerizing factor and depends on constituent stoichiometry. Our findings unravel a sophisticated strategy in which bacterial T3E subverts the host actin cytoskeleton via protein complex coacervation.
| S-EPMC8249405 | biostudies-literature
Project description:Post-transcriptional RNA modifications play an important role in cellular metabolism with homoeostatic disturbances manifesting as a wide repertoire of phenotypes, reduced stress tolerance and translational perturbation, developmental defects, and diseases, such as type II diabetes, leukaemia, and carcinomas. Hence, there has been an intense effort to develop various methods for investigating RNA modifications and their roles in various organisms, including sequencing-based approaches and, more frequently, liquid chromatography-mass spectrometry (LC-MS)-based methods. Although LC-MS offers numerous advantages, such as being highly sensitive and quantitative over a broad detection range, some stationary phase chemistries struggle to resolve positional isomers. Furthermore, the demand for detailed analyses of complex biological samples often necessitates long separation times, hampering sample-to-sample turnover and making multisample analyses time consuming. To overcome this limitation, we have developed an ultra-performance LC-MS (UPLC-MS) method that uses an octadecyl carbon chain (C18)-bonded silica matrix for the efficient separation of 50 modified ribonucleosides, including positional isomers, in a single 9-min sample-to-sample run. To validate the performance and versatility of our method, we analysed tRNA modification patterns of representative microorganisms from each domain of life, namely Archaea (Methanosarcina acetivorans), Bacteria (Pseudomonas syringae), and Eukarya (Saccharomyces cerevisiae). Additionally, our method is flexible and readily applicable for detection and relative quantification using stable isotope labelling and targeted approaches like multiple reaction monitoring (MRM). In conclusion, this method represents a fast and robust tool for broad-range exploration and quantification of ribonucleosides, facilitating future homoeostasis studies of RNA modification in complex biological samples.
| S-EPMC8494288 | biostudies-literature