Analytical Ultracentrifugation Research Articles

Adaptable Self-Assembly of a PEG Dendrimer-Coiled Coil Conjugate.

Self-assembly of designed molecules has enabled the construction of a variety of functional nanostructures. Specifically, adaptable self-assembly has demonstrated several advantageous features for smart materials. Here, we demonstrate that an α-helical coiled coil conjugated with a dendrimer can adapt to spatial restriction due to the strong steric repulsion between dendrimer chains. The adaptable transformation of a tetrameric coiled coil to a trimeric coiled coil can be confirmed using analytical ultracentrifugation upon conjugation of the dendrimer to the coiled coil-forming building block. Interestingly, circular dichroism spectroscopy analysis of the dendrimer conjugate revealed an unconventional trend: the multimerization of the coiled coil is inversely dependent on concentration. This result implies that the spatial crowding between the bulky dendritic chains is significantly stronger than that between linear chains, thereby affecting the overall assembly process. We further illustrated the application potential by decorating the surface of gold nanorods (AuNRs) with the adaptable coiled coil. The dendrimer-coiled coil peptide conjugate can be utilized to fabricate organic-inorganic nanohybrids with enhanced colloidal and thermal stabilities. This study demonstrates that the coiled coil can engage in the adaptable mode of self-assembly with the potential to form dynamic peptide-based materials.

Analytical Ultracentrifugation to Assess the Quality of LNP-mRNA Therapeutics

The approval of safe and effective LNP-mRNA vaccines during the SARS-CoV-2 pandemic is catalyzing the development of the next generation of mRNA therapeutics. Proper characterization methods are crucial for assessing the quality and efficacy of these complex formulations. Here, we show that analytical ultracentrifugation (AUC) can measure, simultaneously and without any sample preparation step, the sedimentation coefficients of both the LNP-mRNA formulation and the mRNA molecules. This allows measuring several quality attributes, such as particle size distribution, encapsulation efficiency and density of the formulation. The technique can also be applied to study the stability of the formulation under stress conditions and different buffers.

Size and Shape Selective Classification of Nanoparticles

As nanoparticle syntheses on a large scale usually yield products with broad size and shape distributions, the properties of nanoparticle-based products need to be tuned after synthesis by narrowing the size and shape distributions or via the removal of undesired fractions. The development of property-selective classification processes requires a universal framework for the quantitative evaluation of multi-dimensional particle fractionation processes. This framework must be applicable to any property and any particle classification process. We extended the well-known one-dimensional methodology commonly used for describing particle size distributions and fractionation processes to the multi-dimensional case to account for the higher complexity of the property distribution and separation functions. In particular, multi-dimensional lognormal distributions are introduced and applied to diameter and length distributions of gold nanorods. The fractionation of nanorods via centrifugation and by orthogonal centrifugal and electric forces is modeled. Moreover, we demonstrate that analytical ultracentrifugation with a multi-wavelength detector (MWL-AUC) is a fast and very accurate method for the measurement of two-dimensional particle size distributions in suspension. The MWL-AUC method is widely applicable to any class of nanoparticles with size-, shape- or composition-dependent optical properties. In addition, we obtained distributions of the lateral diameter and the number of layers of molybdenum disulfide nanosheets via stepwise centrifugation and spectroscopic evaluation of the size fractions.

Methodological validation of sedimentation velocity analytical ultracentrifugation method for Adeno-Associated Virus and collaborative calibration of system suitability substance.

Currently, adeno-associated virus (AAV) is one of the primary gene delivery vectors in gene therapy, facilitating long-term in vivo gene expression. Despite being imperative, it is incredibly challenging to precisely assess AAV particle distribution according to the sedimentation coefficient and identify impurities related to capsid structures. This study performed the systematic methodological validation of quantifying the AAV empty and full capsid ratio. This includes specificity, accuracy, precision, linearity, and parameter variables involving the sedimentation velocity analytical ultracentrifugation (SV-AUC) method. Specifically, SV-AUC differentiated among the empty, partial, full, and High Sedimentation Coefficient Substance (HSCS) AAV particles while evaluating their sedimentation heterogeneity. The intermediate precision analysis of HE (high percentage of empty capsid) and HF( high percentage of full capsid) samples revealed that the specific species percentage, such as empty or full, was more significant than 50%. Moreover, the RSD (relative standard deviation) could be within 5%. Even for empty or partial less than 15%, the RSD could be within 10%. The accuracy recovery rates of empty capsid were between 103.9% and 108.7% across three different mixtures. When the measured percentage of specific species was more significant than 14%, the recovery rate was between 77.9% and 106.6%. Linearity analysis revealed an excellent linear correlation between the empty, partial, and full in the HE samples. The AAV samples with as low as 7.4×1011 cp/mL AAV could be accurately quantified with SV-AUC. The parameter variable analyses revealed that variations in cell alignment significantly affected the overall results. Still, the detection wavelength of 235nm slightly influenced the empty, partial, and full percentages. Minor detection wavelength changes showed no impact on the sedimentation coefficient of these species. However, the temperature affected the measured sedimentation coefficient. These results validated the SV-AUC method to quantify AAV. This study provides solutions to AAV empty and full capsid ratio quantification challenges and the subsequent basis for calibrating the AAV empty capsid system suitability substance. Due to the AAV structure and potential variability complexity in detection, we jointly calibrated empty capsid system suitability substance with three laboratories to accurately detect the quantitative AAV empty and full capsid ratio. The empty capsid system suitability substance could be used as an external reference to measure the performance of the instrument. The results could be compared with multiple QC (quality control)laboratories based on the AAV vector and calibration accuracy. This is crucial for AUC to be used for QC release and promote gene therapy research worldwide.

Phosphorylation in the Ser/Arg-rich region of the nucleocapsid of SARS-CoV-2 regulates phase separation by inhibiting self-association of a distant helix

The nucleocapsid protein (N) of SARS-CoV-2 is essential for virus replication, genome packaging, evading host immunity, and virus maturation. N is a multidomain protein composed of an independently folded monomeric N-terminal domain that is the primary site for RNA binding, and a dimeric C-terminal domain that is essential for efficient phase separation and condensate formation with RNA. The domains are separated by a disordered Ser/Arg-rich region preceding a self-associating Leu-rich helix. Phosphorylation in the Ser/Arg region in infected cells decreases the viscosity of N:RNA condensates promoting viral replication and host immune evasion. The molecular level effect of phosphorylation, however, is missing from our current understanding. Using NMR spectroscopy and analytical ultracentrifugation we show that phosphorylation destabilizes the self-associating Leu-rich helix 30 amino acids distant from the phosphorylation site. NMR and gel shift assays demonstrate that RNA binding by the linker is dampened by phosphorylation, whereas RNA binding to the full-length protein is not significantly affected presumably due to retained strong interactions with the primary RNA binding domain. Introducing a switchable self-associating domain to replace the Leu-rich helix confirms the importance of linker self-association to droplet formation and suggests that phosphorylation not only increases solubility of the positively charged elongated Ser/Arg region as observed in other RNA binding proteins but can also inhibit self-association of the Leu-rich helix. These data highlight the effect of phosphorylation both at local sites and at a distant self-associating hydrophobic helix in regulating liquid-liquid phase separation of the entire protein.

Challenges in the analysis of pharmaceutical lentiviral vector products by orthogonal and complementary physical (nano)particle characterization techniques

Lentiviral vectors (LVVs) are used as a starting material to generate chimeric antigen receptor (CAR) T cells. Therefore, LVVs need to be carefully analyzed to ensure safety, quality, and potency of the final product. We evaluated orthogonal and complementary analytical techniques for their suitability to characterize particulate matter (impurities and LVVs) in pharmaceutical LVV materials at development stage derived from suspension and adherent manufacturing processes. Microfluidic resistive pulse sensing (MRPS) with additional manual data fitting enabled the assessment of mode diameters for particles in the expected LVV size range in material from adherent production. LVV material from a suspension process, however, contained substantial amounts of particulate impurities which blocked MRPS cartridges. Sedimentation-velocity analytical ultracentrifugation (SV-AUC) resolved the LVV peak in material from adherent production well, whereas in more polydisperse samples from suspension production, presence of particulate impurities masked a potential signal assignable to LVVs. In interferometric light microscopy (ILM) and nanoparticle tracking analysis (NTA), lower size detection limits close to ∼ 70 nm resulted in an apparent peak in particle size distributions at the expected size for LVVs emphasizing the need to interpret these data with care. Interpretation of data from dynamic light scattering (DLS) was limited by insufficient size resolution and sample polydispersity. In conclusion, the analysis of LVV products manufactured at pharmaceutical scale with current state-of-the-art physical (nano)particle characterization techniques was challenging due to the presence of particulate impurities of heterogeneous size. Among the evaluated techniques, MRPS and SV-AUC were most promising yielding acceptable results at least for material from adherent production.

Biochemical, structural, and computational analyses of two new clinically identified missense mutations of ALDH7A1

Aldehyde dehydrogenase 7A1 (ALDH7A1) catalyzes a step of lysine catabolism. Certain missense mutations in the ALDH7A1 gene cause pyridoxine dependent epilepsy (PDE), a rare autosomal neurometabolic disorder with recessive inheritance that affects almost 1:65,000 live births and is classically characterized by recurrent seizures from the neonatal period. We report a biochemical, structural, and computational study of two novel ALDH7A1 missense mutations that were identified in a child with rare recurrent seizures from the third month of life. The mutations affect two residues in the oligomer interfaces of ALDH7A1, Arg134 and Arg441 (Arg162 and Arg469 in the HGVS nomenclature). The corresponding enzyme variants R134S and R441C (p.Arg162Ser and p.Arg469Cys in the HGVS nomenclature) were expressed in Escherichia coli and purified. R134S and R441C have 10,000- and 50-fold lower catalytic efficiency than wild-type ALDH7A1, respectively. Sedimentation velocity analytical ultracentrifugation shows that R134S is defective in tetramerization, remaining locked in a dimeric state even in the presence of the tetramer-inducing coenzyme NAD+. Because the tetramer is the active form of ALDH7A1, the defect in oligomerization explains the very low catalytic activity of R134S. In contrast, R441C exhibits wild-type oligomerization behavior, and the 2.0 Å resolution crystal structure of R441C complexed with NAD+ revealed no obvious structural perturbations when compared to the wild-type enzyme structure. Molecular dynamics simulations suggest that the mutation of Arg441 to Cys may increase intersubunit ion pairs and alter the dynamics of the active site gate. Our biochemical, structural, and computational data on two novel clinical variants of ALDH7A1 add to the complexity of the molecular determinants underlying pyridoxine dependent epilepsy.

Quantitation of Enterovirus A71 Empty and Full Particles by Sedimentation Velocity Analytical Ultracentrifugation.

The enterovirus A71 (EV71) inactivated vaccine is an effective intervention to control the spread of the virus and prevent EV71-associated hand, foot, and mouth disease (HFMD). It is widely administered to infants and children in China. The empty particles (EPs) and full particles (FPs) generated during production have different antigenic and immunogenic properties. However, the antigen detection methods currently used were established without considering the differences in antigenicity between EPs and FPs. There is also a lack of other effective analytical methods for detecting the different particle forms, which hinders the consistency between batches of products. In this study, we analyzed the application of sedimentation velocity analytical ultracentrifugation (SV-AUC) in characterizing the EPs and FPs of EV71. Our results showed that the proportions of the two forms could be quantified simultaneously by SV-AUC. We also determined the repeatability and accuracy of this method and found that both parameters were satisfactory. We assessed SV-AUC for bulk vaccine quality control, and our findings indicated that SV-AUC can be used effectively to analyze the percentage of EPs and FPs and monitor the consistency of the process to ensure the quality of the vaccine.

Design and Escherichia coli Expression of a Natively Folded Multi-Disulfide Bonded Influenza H1N1-PR8 Receptor-Binding Domain (RBD).

Refolding multi-disulfide bonded proteins expressed in E. coli into their native structure is challenging. Nevertheless, because of its cost-effectiveness, handiness, and versatility, the E. coli expression of viral envelope proteins, such as the RBD (Receptor-Binding Domain) of the influenza Hemagglutinin protein, could significantly advance research on viral infections. Here, we show that H1N1-PR8-RBD (27 kDa, containing four cysteines forming two disulfide bonds) expressed in E. coli and was purified with nickel affinity chromatography, and reversed-phase HPLC was successfully refolded into its native structure, as assessed with several biophysical and biochemical techniques. Analytical ultracentrifugation indicated that H1N1-PR8-RBD was monomeric with a hydrodynamic radius of 2.5 nm. Thermal denaturation, monitored with DSC and CD at a wavelength of 222 nm, was cooperative with a midpoint temperature around 55 °C, strongly indicating a natively folded protein. In addition, the 15N-HSQC NMR spectrum exhibited several 1H-15N resonances indicative of a beta-sheeted protein. Our results indicate that a significant amount (40 mg/L) of pure and native H1N1-PR8-RBD can be produced using an E. coli expression system with our refolding procedure, offering potential insights into the molecular characterization of influenza virus infection.

Identification, Characterization, Cloning, and Cross-Reactivity of Zan b 2, a Novel Pepper Allergen of 11S Legumin.

Protein from Sichuan peppers can elicit mild to severe allergic reactions. However, little is known about their allergenic proteins. We aimed to isolate, identify, clone, and characterize Sichuan pepper allergens and to determine its allergenicity and cross-reactivities. Sichuan pepper seed proteins were extracted and then analyzed by SDS-PAGE. Western blotting was performed with sera from Sichuan pepper-allergic individuals. Proteins of interest were purified using hydrophobic interaction chromatography and gel filtration and further analyzed by analytical ultracentrifugation, circular dichroism spectroscopy, and mass spectrometry (MS). Their coding region was amplified in the genome. IgE reactivity and cross-reactivity of allergens were evaluated by dot blot, enzyme-linked immunosorbent assay (ELISA), and competitive ELISA. Western blot showed IgE binding to a 55 kDa protein. This protein was homologous to the citrus proteins and has high stability and a sheet structure. Four DNA sequences were cloned. Six patients' sera (60%) showed specific IgE reactivity to this purified 11S protein, which was proved to have cross-reactivation with extracts of cashew nuts, pistachios, and citrus seeds. A novel allergen in Sichuan pepper seeds, Zan b 2, which belongs to the 11S globulin family, was isolated and identified. Its cross-reactivity with cashew nuts, pistachios, and citrus seeds was demonstrated.

Abstract 7578: BRD4 protein degradation for p53 mutant diffuse intrinsic pontine glioma (DIPG) via various delivery approaches for ARV-825 by using camel milk exosomes

Abstract DIPGs (diffuse intrinsic pontine gliomas) are a particularly aggressive and malignant type of pediatric brain tumor. Given the tumor's location, surgery has a restricted role, and it is insensitive to most chemotherapies. Although fractionated radiation is the standard of therapy for DIPG, the cancer invariably worsens in 6-12 months. TP53 tumor suppressor protein mutations are found in 70-80% of DIPG tumors. These TP53 mutations have been linked to radiation resistance in DIPG patients. Our goal was to find new pharmacological compounds that would improve the radiation sensitivity of p53 mutant DIPG cell lines and to determine their molecular mechanism of action. Numerous bromodomain-containing protein 4 (BRD4) inhibitors have entered clinical trials in recent years, yielding promising outcomes in tumor treatment since BRD4 expression in DIPG is substantially higher than in neighboring normal brain tissue. So consequently, BRD4 is a target for DIPG treatment. ARV-825 is an emerging PROTAC (Proteolysis Targeting Chimera) compound which can cause rapid and persistent BRD4 protein degradation. However, delivering ARV-825 to pons is a major challenge owing to poor oral bioavailability of PROTACS. In this study, we used camel milk derived exosomes (CME) for delivery of ARV-825 to pons by both nasal and oral routes. To begin with, goat milk exosomes were also considered but CME was chosen because of its innate cytotoxicity against DIPG cell lines. CME were isolated using ultracentrifugation and nanoparticle tracking analysis and revealed a size distribution of 112.5 ± 2.8 nm, zeta potential of -26.38 ± 0.12 mV. CME (1*1011) were cytotoxic to both SF8628 (with a H3.3 K27M mutation) and SF188 (grade IV pediatric glioblastoma with wild type H3.3) cell lines and had 51% and 44% cell viability respectively. ARV-825 showed IC50 of 546.58 ± 22.14 nM and 608.94 ± 30.04 nM in SF8628 cells and SF188 cells respectively. ARV-825’s IC50 was lowered when ARV-825 loaded CME were used. ARV-825 was loaded using sonication and entrapment efficiency was found to be 42.96 ± 2.14 % by HPLC analysis. CME were labelled with Vybrant DiO (CME-DIO) and instilled into each nostril of Sprague Dawley rats daily. Brain sections were viewed with fluorescent microscope and CME-DIO were seen in olfactory region after 3 days and in the pons region after 5 days demonstrating the ability of CME to reach the target site. In-vitro permeability studies using MDCK cells showed approximately 9µg/mL (60%), 15µg/mL (80%) and 16µg/mL (100%) of ARV-825 permeation in 2, 4 and 24 hours respectively when loaded into CME. The results of the present study suggest the possibility of using CME as a viable delivery strategy for ARV-825 against DIPG. Further in vitro and in vivo studies are currently undergoing to validate the results and understand kinetics and the molecular mechanisms of ARV-825 loaded CME. Citation Format: Aakash Nathani, Mounika Aare, Li Sun, Yan Li, Mandip Singh. BRD4 protein degradation for p53 mutant diffuse intrinsic pontine glioma (DIPG) via various delivery approaches for ARV-825 by using camel milk exosomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7578.

Abstract 1346: Combined role of interleukin-15 stimulated natural killer cell-derived extracellular vesicles and carboplatin in Osimertinib resistant H1975 lung cancer with EGFR mutations

Abstract Extracellular vesicles (EVs) derived from immune cells (e.g., NK cells, dendritic cells) may be able to treat malignancies that are resistant to other treatments (e.g., chemotherapy) relying on the hypothesis that EVs carry almost the same cargo of the parent cells. In this study, we evaluated IL-15 stimulated natural killer cell-derived EVs (NK-EVs) as therapeutic agents in vitro and in vivo in Osimertinib resistant lung cancer (H1975R) with EGFR mutations(L858R). NK-EVs were isolated by ultracentrifugation and nanoparticle tracking analysis revealed a size distribution of 89.5 ± 3.4 nm, zeta potential of -31.38 ± 0.25 mV. Atomic force microscopy imaging revealed vesicles with a spherical form and comparable sizes, meeting the criteria of exosomal EVs. Further, western blot studies demonstrated the presence of regular EV markers along with specific NK markers (perforin and granzyme). EVs were also characterized by using proteomic analysis which demonstrated that EVs had proteins for natural killer cell mediated cytotoxicity (Granzyme B) and T cell activation (Perforin and Plastin-2). Gene oncology (GO) analysis also showed that these are differentially expressed proteins (DEPs) that are involved in programmed cell death and positive regulation of cell death. Further, isolated NK-EVs (1*1011 particles/mL) were cytotoxic to H1975R cells in vitro with 55% and 68% cell viability in 2D and 3D cell cultures respectively against control. Carboplatin’s IC50 was reduced by approximately 1.9 and 1.7-fold (p less than 0.001) in 2D and 3D cell culture respectively when combined with NK-EVs. The EVs were then combined with carboplatin (25 mg/kg) and administered by i.p. route to H1975R tumor xenografts and significant reduction in tumor volume in vivo (≈1000 mm3 compared to ≈2000 mm3 of control group, p less than 0.001) was observed after 10 days. Our findings show for the first time that NK-EVs target the PD-L1/PD-1 immunological checkpoint and induce apoptosis and anti-inflammatory response by downregulation of SOD2, PARP, BCL2, SET, NF-κB and TGF-ß. MicroRNAs regulating cytotoxic proteins like perforin, granzyme and plastin were also identified using sequencing and the miRNAs: hsa-miR-5193/149-5P/3133/193-5p are picked for further studies. These miRNAs have been encapsulated in NK-EVs by electroporation and will be evaluated against Lung PDX xenografts along with carboplatin loaded NK-EVs. These miRNAs are directly involved in T cell or NK cell mediated cytotoxicity and are being currently investigated. The ability to isolate functional NK-EVs on a large scale and using them with platinum-based drugs may lead to new clinical applications. The results of the present study suggest the possibility of the combination of NK-cell-derived EVs and carboplatin as a viable immunochemotherapeutic strategy for resistant cancers. Citation Format: Aakash Nathani, Li Sun, Islauddin Khan, Mounika Aare, Arvind Bagde, Yan Li, Mandip Sachdeva. Combined role of interleukin-15 stimulated natural killer cell-derived extracellular vesicles and carboplatin in Osimertinib resistant H1975 lung cancer with EGFR mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1346.

Structure of native four-repeat satellite III sequence with non-canonical base interactions.

Tandem-repetitive DNA (where two or more DNA bases are repeated numerous times) can adopt non-canonical secondary structures. Many of these structures are implicated in important biological processes. Human Satellite III (HSat3) is enriched for tandem repeats of the sequence ATGGA and is located in pericentromeric heterochromatin in many human chromosomes. Here, we investigate the secondary structure of the four-repeat HSat3 sequence 5'-ATGGAATGGA ATGGA ATGGA-3' using X-ray crystallography, NMR, and biophysical methods. Circular dichroism spectroscopy, thermal stability, native PAGE, and analytical ultracentrifugation indicate that this sequence folds into a monomolecular hairpin with non-canonical base pairing and B-DNA characteristics at concentrations below 0.9 mM. NMR studies at 0.05-0.5 mM indicate that the hairpin is likely folded-over into a compact structure with high dynamics. Crystallographic studies at 2.5 mM reveal an antiparallel self-complementary duplex with the same base pairing as in the hairpin, extended into an infinite polymer. The non-canonical base pairing includes a G-G intercalation sandwiched by sheared A-G base pairs, leading to a cross-strand four guanine stack, so called guanine zipper. The guanine zippers are spaced throughout the structure by A-T/T-A base pairs. Our findings lend further insight into recurring structural motifs associated with the HSat3 and their potential biological functions.

BASIS: BioAnalysis SEDFIT integrated software for cGMP analysis of SV-AUC data.

Sedimentation velocity analytical ultracentrifugation (SV-AUC) has long been an important method for characterization of antibody therapeutics. Recently, SV-AUC has experienced a wave of new interest and usage from the gene and cell therapy industry, where SV-AUC has proven itself to be the "gold standard" analytical approach for determining capsid loading ratios for adeno-associated virus (AAV) and other viral vectors. While other more common approaches have existed in the realm of cGMP-compliant techniques for years, SV-AUC has long been used strictly for characterization, but not for release testing. This manuscript describes the challenges faced in bringing SV-AUC to a cGMP environment and describes a new program, "BASIS", which allows for 21 CFR Part 11-compliant data handling and data analysis using the well-known and frequently cited SEDFIT analysis software.

Methods for the Design and Analysis of Analytical Ultracentrifugation Experiments.

Analytical ultracentrifugation experiments play an integral role in the solution-phase characterization of biological macromolecules and their interactions. This unit discusses the design of sedimentation velocity and sedimentation equilibrium experiments performed with a Beckman Proteomelab XL-A or XL-I analytical ultracentrifuge and with a Beckman Optima AUC. Instrument settings and experimental design considerations are explained, and strategies for the analysis of experimental data with the UltraScan data analysis software package are presented. Special attention is paid to the strengths and weaknesses of the available detectors, and guidance is provided on how to extract maximum information from analytical ultracentrifugation experiments. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC.

Versatile Capillary Cells for Handling Concentrated Samples in Analytical Ultracentrifugation.

In concentrated macromolecular dispersions, far-from-ideal intermolecular interactions determine the dispersion behaviors including phase transition, crystallization, and liquid-liquid phase separation. Here, we present a novel versatile capillary-cell design for analytical ultracentrifugation-sedimentation equilibrium (AUC-SE), ideal for studying samples at high concentrations. Current setups for such studies are difficult and unreliable to handle, leading to a low experimental success rate. The design presented here is easy to use, robust, and reusable for samples in both aqueous and organic solvents while requiring no special tools or chemical modification of AUC cells. The key and unique feature is the fabrication of liquid reservoirs directly on the bottom window of AUC cells, which can be easily realized by laser ablation or mechanical drilling. The channel length and optical path length are therefore tunable. The success rate for assembling this new cell is close to 100%. We demonstrate the practicality of this cell by studying: (1) the equation of state and second virial coefficients of concentrated gold nanoparticle dispersions in water and bovine serum albumin (BSA) as well as lysozyme solution in aqueous buffers, (2) the gelation phase transition of DNA and BSA solutions, and (3) liquid-liquid phase separation of concentrated BSA/polyethylene glycol (PEG) droplets.

Flexible structural arrangement and DNA-binding properties of protein p6 from Bacillus subtillis phage φ29.

The genome-organizing protein p6 of Bacillus subtilis bacteriophage φ29 plays an essential role in viral development by activating the initiation of DNA replication and participating in the early-to-late transcriptional switch. These activities require the formation of a nucleoprotein complex in which the DNA adopts a right-handed superhelix wrapping around a multimeric p6 scaffold, restraining positive supercoiling and compacting the viral genome. Due to the absence of homologous structures, prior attempts to unveil p6's structural architecture failed. Here, we employed AlphaFold2 to engineer rational p6 constructs yielding crystals for three-dimensional structure determination. Our findings reveal a novel fold adopted by p6 that sheds light on its self-association mechanism and its interaction with DNA. By means of protein-DNA docking and molecular dynamic simulations, we have generated a comprehensive structural model for the nucleoprotein complex that consistently aligns with its established biochemical and thermodynamic parameters. Besides, through analytical ultracentrifugation, we have confirmed the hydrodynamic properties of the nucleocomplex, further validating in solution our proposed model. Importantly, the disclosed structure not only provides a highly accurate explanation for previously experimental data accumulated over decades, but also enhances our holistic understanding of the structural and functional attributes of protein p6 during φ29 infection.

Organic Nucleation: Water Rearrangement Reveals the Pathway of Ibuprofen.

The organic nucleation of the pharmaceutical ibuprofen is investigated, as triggered by the protonation of ibuprofen sodium salt at elevated pH. The growth and aggregation of nanoscale solution species by Analytical Ultracentrifugation and Molecular Dynamics (MD) simulations is tracked. Both approaches reveal solvated molecules, oligomers, and prenucleation clusters, their size as well as their hydration at different reaction stages. By combining surface-specific vibrational spectroscopy and MD simulations, water interacting with ibuprofen at the air-water interface during nucleation is probed. The results show the structure of water changes upon ibuprofen protonation in response to the charge neutralization. Remarkably, the water structure continues to evolve despite the saturation of protonated ibuprofen at the hydrophobic interface. This further water rearrangement is associated with the formation of larger aggregates of ibuprofen molecules at a late prenucleation stage. The nucleation of ibuprofen involves ibuprofen protonation and their hydrophobic assembly. The results highlight that these processes are accompanied by substantial water reorganization. The critical role of water is possibly relevant for organic nucleation in aqueous environments in general.

Dimerization of the CNNM extracellular domain.

Cystathionine- -synthase (CBS)-pair domain divalent metal cation transport mediators (CNNMs) are an evolutionarily conserved family of magnesium transporters. They mediate magnesium homeostasis directly by transport of Mg2+ ions and indirectly by regulation of the transient receptor potential ion channel subfamily M member 7 (TRPM7). Here, we report the crystal structure of the extracellular domain of tapeworm CNNM4. The domain forms a dimer of immunoglobulin-like (Ig-like) folds with electron density observed for three glycosylation sites. Analytical ultracentrifugation confirms that mutations in the extracellular domain of human CNNM4 prevent its dimerization. An analogous mutation in mouse CNNM2 impairs its activity in a cellular assay of Mg2+ transport.

Complexation of poly(methacrylic acid) star polyelectrolytes with lysozyme

Protein-polyelectrolyte complexes are of great interest due to their applications in both biological and industrial systems. This study focuses on investigating the electrostatic complexation between (hen egg white) lysozyme (LYZ), a well-studied enzyme, and two star-shaped poly(methacrylic acid) (PMAA) polymers with different arm molar masses, namely S-PMAA5K and S-PMAA10K. Depending on the mixing ratio of the two components (protein/star polyelectrolyte) either stable solutions were formed or macroscopic phase separation, i.e. coacervation, was observed. Dynamic and electrophoretic light scattering results reveal that the overall physicochemical properties of the complexes and the strength of the interaction depends on the ratio of the two components as well as on the molar mass of the polymer, with the S-PMAA10K series exhibiting stronger interactions compared to S-PMAA5K. Additional information regarding the molar mass of the star polyelectrolytes and the corresponding complexes with lysozyme were acquired by analytical ultracentrifugation. The morphology of the complexes was further elucidated through atomic force and cryo-transmission electron microscopy showing globular raspberry-like objects. Moreover, fluorescence and infrared spectroscopic techniques were employed to explore potential conformational changes of the protein upon complexation, with both techniques indicating that the protein does not undergo significant structural changes. Furthermore, UV–Vis spectroscopy was utilized to quantify the amount of protein present in the supernatant of the coacervated complex solutions, thus allowing the quantification of the unbound protein remaining in the solution after complex formation.