Protein charge parameters that influence stability and cellular internalization of polyelectrolyte complex micelles

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, 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 as well as the importance of correlating solution structure and desired functional activity.

‘Get in touch with yourself’: The Structure of Relationship Advice in Women’s Magazines

This study investigates the way sex and relationship advice articles are structured in four English language women’s magazines. Cosmopolitan and Marie Claire were selected from the US, and Female and Her World from Malaysia. Forty articles were selected for the analysis. The study adopts Machin and Van Leeuwen’s (2003) problem-solution structure, besides using discourse pragmatic analysis. By studying this genre in the two different contexts, one of the main things that emerged is that this particular genre is more complex and diverse than what other researchers have found. The writers of the advice resort to various strategies and techniques to attract women to read these articles. They also have to balance social and cultural sensitivities with their message of freedom and liberation for women as appeared in the Malaysian data. Thus, studying this genre gives useful insights on how culture affects the texts and vice-versa.

Mapping the Deformability of Natural and Designed Cellulosomes in solution

Background : Natural cellulosome multi-enzyme complexes, their components, and engineered ‘designer cellulosomes’ (DCs) promise an efficient means of breaking down cellulosic substrates into valuable biofuel products. Their broad uptake in biotechnology relies on boosting proximity-based synergy among the resident enzymes but the modular architecture challenges structure determination and rational design.Results: We used small angle X-ray scattering combined with molecular modeling to study the solution structure of cellulosomal components. These include three dockerin-bearing cellulases with distinct substrate specificities, original scaffoldins from the human gut bacterium Ruminococcus champanellensis (ScaA, ScaH and ScaK) and a trivalent cohesin-bearing designer scaffoldin (Scaf20L), followed by cellulosomal complexes comprising these components, and the nonavalent fully loaded Clostridium thermocellum CipA in complex with Cel8A from the same bacterium. The size analysis of Rg and Dmax values deduced from the scattering curves and corresponding molecular models highlight their variable aspects, depending on composition, size and spatial organization of the objects in solution.Conclusion: Our data quantifies variability of form and compactness of cellulosomal components in water and confirms that this native plasticity may well be related to speciation with respect to the substrate that is targeted. By showing that scaffoldins or components display enhanced compactness compared to the free objects, we provide new routes to rationally enhance their stability and performance in their environment of action.

The PNUTS-PAD domain recruits MYC to the PNUTS:PP1 phosphatase complex via the oncogenic MYC-MB0 region

SummaryDespite MYC dysregulation in most human cancers, strategies to target this potent oncogenic driver remains an urgent unmet need. Recent evidence shows the PP1 phosphatase and its regulatory subunit PNUTS control MYC phosphorylation and stability, however the molecular basis remains unclear. Here we demonstrate that MYC interacts directly with PNUTS through the MYC homology Box 0 (MB0), a highly conserved region recently shown to be important for MYC oncogenic activity. MB0 interacts with PNUTS residues 1-148, a functional unit here termed, PNUTS amino-terminal domain (PAD). Using NMR spectroscopy we determined the solution structure of PAD, and characterised its interaction with MYC. Point mutations of residues at the MYC-PNUTS interface significantly weaken their interaction both in vitro and in vivo. These data demonstrate the MB0 binding pocket of the PAD represents an attractive site for pharmacological disruption of the MYC-PNUTS interaction.In BriefSolving the structure of MYC-PNUTS direct interaction reveals how the intrinsically disordered MYC-Box0 (MB0) region anchors into a binding pocket in the N-terminal PAD domain of PNUTS. These data provide insight into the molecular mechanism of how the PNUTS:PP1 phosphatase complex regulates MYC phosphorylation.HighlightsA region critical for MYC oncogenesis, MYC-Box0 (MB0), directly interacts with PNUTSPNUTS amino-terminal domain (PAD) is a structural domain that interacts with MYC MB0Mutation of single residues at the interaction interface disrupts MYC-PNUTS binding in cellsMYC-PNUTS binding releases MYC intramolecular interactions to enable PP1substrate access

Mapping the electrostatic potential of the nucleosome acidic patch

The nucleosome surface contains an area with negative electrostatic potential known as the acidic patch, which functions as a binding platform for various proteins to regulate chromatin biology. The dense clustering of acidic residues may impact their effective pKa and thus the electronegativity of the acidic patch, which in turn could influence nucleosome-protein interactions. We here set out to determine the pKa values of residues in and around the acidic patch in the free H2A-H2B dimer using NMR spectroscopy. We present a refined solution structure of the H2A-H2B dimer based on intermolecular distance restraints, displaying a well-defined histone-fold core. We show that the conserved histidines H2B H46 and H106 that line the acidic patch have pKa of 5.9 and 6.5, respectively, and that most acidic patch carboxyl groups have pKa values well below 5.0. For H2A D89 we find strong evidence for an elevated pKa of 5.3. Our data establish that the acidic patch is highly negatively charged at physiological pH, while protonation of H2B H106 and H2B H46 at slightly acidic pH will reduce electronegativity. These results will be valuable to understand the impact of pH changes on nucleosome-protein interactions in vitro, in silico or in vivo.

Probing the existence of non-thermal Terahertz radiation induced changes of the protein solution structure

During the last decades discussions were taking place on the existence of global, non-thermal structural changes in biological macromolecules induced by Terahertz (THz) radiation. Despite numerous studies, a clear experimental proof of this effect for biological particles in solution is still missing. We developed a setup combining THz-irradiation with small angle X-ray scattering (SAXS), which is a sensitive method for detecting the expected structural changes. We investigated in detail protein systems with different shape morphologies (bovine serum albumin, microtubules), which have been proposed to be susceptible to THz-radiation, under variable parameters (THz wavelength, THz power densities up to 6.8 mW/cm2, protein concentrations). None of the studied systems and conditions revealed structural changes detectable by SAXS suggesting that the expected non-thermal THz-induced effects do not lead to alterations of the overall structures, which are revealed by scattering from dissolved macromolecules. This leaves us with the conclusion that, if such effects are present, these are either local or outside of the spectrum and power range covered by the present study.

High Selectivity and Reusability of Biomass-Based Adsorbent for Chloramphenicol Removal

Recently, biomass-based materials have attracted increasing attention because of their advantages of low cost, environment-friendly and nonpollution. Herein, the feasibility of using corn stalk biomass fiber (CF) and Fe3O4 embedded chitosan (CS) as a novel biomass-based adsorbent (CFS) to remove chloramphenicol (CAPC) from aqueous solution. Structure of CFS was characterized by using X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Brunauer–Emmett–Teller (BET), scanning electron microscopy (SEM) and zeta potential techniques. The effects of solution pH, adsorption time and ion strength on the adsorption capacity were examined. Adsorption isotherms obtained from batch experiments were better fitted by Langmuir model compared with Freundlich model, Dubinin–Radushkevich model and Temkin model. Adsorption kinetic data matched well to the pseudo-second order kinetic model. CAPC adsorption was endothermic, spontaneous, and entropy-increasing nature on CFS. In addition, the CFS could be separated by an external magnetic field, recycled, and reused without any significant loss in the adsorption capacity of CAPC. Based on these excellent performances, there is potential that CFS can be considered as a proficient and economically suitable material for the CAPC removal from the water environment.