Analytical Ultracentrifugation Research Articles

End Plate Chondrocyte-Derived Exosomal miR-133a-3p Alleviates Intervertebral Disc Degeneration by Targeting the NF-κB Signaling Pathway through the miR-133a-3p/MAML1 Axis.

Chondrocyte-derived exosomes have shown efficacy in differentiating osteoarthritis-affected cartilage. Intervertebral disc degeneration (IVDD) and osteoarthritis often affect facet joints of the spine and show common epidemiological and pathophysiological characteristics. However, the potential of chondrocyte-derived exosomes for treating IVDD remains unclear. The present study aimed to confirm the effect of end plate chondrocyte-derived exosomes (EPC-Exo) on IVDD and elucidate the underlying mechanism. EPC-Exos were isolated and identified by ultracentrifugation, Western blotting, electron microscopy, and nanoparticle tracking analysis. In the in vitro, EPC-Exo uptake by nucleus pulposus (NP) cells reduced cell death by blocking the nuclear factor-κB (NF-κB) signaling pathway. In the in vivo study, EPC-Exos injected into rat intervertebral discs mitigated lipopolysaccharide-induced IVDD, as revealed by a decreased loss of disc height and improved magnetic resonance imaging findings and histological scores. Bioinformatics and sequencing analyses indicated that EPC-Exos alleviated IVDD through the miR-133a-3p/MAML1 axis. The present study suggests that EPC-Exos reduced IVDD incidence via the miR-133a-3p/MAML1 axis-mediated suppression of NF-κB signaling, which prevented the pyroptosis of NP cells.

Solution conformational differences between conventional and CENP-A nucleosomes are accentuated by reversible deformation under high pressure.

Solution-based interrogation of the physical nature of nucleosomes has its roots in X-ray and neutron scattering experiments, including those that provided the initial observation that DNA wraps around core histones. In this study, we performed a comprehensive small-angle scattering study to compare canonical nucleosomes with variant centromeric nucleosomes harboring the histone variant, CENP-A. We used nucleosome core particles (NCPs) assembled on an artificial positioning sequence (Widom 601) and compared these to those assembled on a natural α-satellite DNA cloned from human centromeres. We establish the native solution properties of octameric H3 and CENP-A NCPs using analytical ultracentrifugation (AUC), small-angle X-ray scattering (SAXS), and contrast variation small-angle neutron scattering (CV-SANS). Using high-pressure SAXS (HP-SAXS), we discovered that both histone identity and DNA sequence have an impact on the stability of octameric nucleosomes in solution under high pressure (300 MPa), with evidence of reversible unwrapping in these experimental conditions. Both canonical nucleosomes harboring conventional histone H3 and their centromeric counterparts harboring CENP-A have a substantial increase in their radius of gyration, but this increase is much less prominent for centromeric nucleosomes. More broadly for chromosome-related research, we note that as HP-SAXS methodologies expand in their utility, we anticipate this will provide a powerful solution-based approach to study nucleosomes and higher-order chromatin complexes.

Comparative analysis of empty and full adeno-associated viruses under stress conditions by anion-exchange chromatography, analytical ultracentrifugation, and mass photometry.

Adeno-associated viruses (AAVs) have emerged as a promising gene delivery vehicle for the treatment of diseases. As AAVs are a complex therapeutic modality, new analytical techniques are needed to thoroughly characterize the critical quality attributes (CQAs) to support drug development. Empty and full ratio is one of the CQAs of AAVs that may impact drug safety and efficacy. While the empty and full ratio of AAV therapeutics in untreated conditions can be well characterized by different analytical methods, limited studies have demonstrated whether these analytical methods can be used for characterizing stressed AAVs, which can help with assessing the stability of the molecule and identify potential degradation pathways. Here, we employ three orthogonal analytical techniques - (1) anion-exchange chromatography (AEX), (2) analytical ultracentrifugation (AUC), and (3) recently introduced mass photometry (MP) - to investigate their ability to characterize AAV samples subjected to various stress conditions, including freeze/thaw, physical agitation, and thermal stress. Based on our observations, AEX is a high-throughput technique. However, it falls short in quantifying the amount of partially filled AAVs, and the quantification is significantly impacted by post-translational modifications, which often occur to AAVs under stress conditions. AUC provides the best resolution for stressed AAV samples but is limited by its throughput and high sample consumption. MP combines the strength of being high-throughput and requires the least amount of samples, albeit at the expense of lower resolution compared to AUC. Our study suggests that each of these three techniques, AEX, AUC, and the emerging MP method, is suitable for characterizing empty and full AAV particles at various stages throughout the lifecycle of drug development.

Analysis of Giant-Shell CdSe/CdS Quantum Dots via Analytical Ultracentrifugation Combined with Spectrally Resolved Photoluminescence.

Knowledge of the structure-property relationships of functional nanomaterials, including, for example, their size- and composition-dependent photoluminescence (PL) and particle-to-particle variations, is crucial for their design and reproducibility. Herein, the Angstrom-resolution capability of an analytical ultracentrifuge combined with an in-line multiwavelength emission detection system (MWE-AUC) for measuring the sedimentation coefficient-resolved spectrally corrected PL spectra of dispersed nanoparticles is demonstrated. The capabilities of this technique are shown for giant-shell CdSe/CdS quantum dots (g-QDs) with a PL quantum yield (PL QY) close to unity capped with oleic acid and oleylamine ligands. The MWE-AUC PL measurements are calibrated and validated with certified fluorescence standards. The spectrally corrected and size-dependent PL spectra of the g-QDs derived from a single MWE-AUC experiment are then analyzed and compared with the results of single-particle spectroscopic studies, yielding the PL spectra, decay kinetics, and blinking behavior of individual g-QDs. This study underlines the vast potential of MWE-AUC with in-line optical detection for the characterization of advanced nanomaterials with a complex structure.

When conventional approach in toxicity assays falls short for nanomedicines: a case study with nanoemulsions.

The aim of this study was to assess the critical quality attributes of parenteral nanoemulsion formulations by measuring several physicochemical parameters and linking them to their in vitro performance, illustrating how simplistic and routinely used approaches are insufficient for understanding a potential nanomedicine. Physicochemical characterization should encompass size and size distribution through at least two orthogonal techniques, such as dynamic light scattering (DLS) and electron microscopy, with added value from analytical ultracentrifugation. In vitro toxicity assessment was performed using three different assays to determine mitochondrial activity (WST-1), membrane integrity (lactate dehydrogenase release (LDH) assay), and cell viability (propidium iodide (PI) staining). Special focus was placed on estimating appropriate incubation times for relevant results in biological investigations. All formulations had an average diameter of around 100nm. Conclusions regarding in vitro safety were assay-dependent: LDH and PI-based assays showed good correlation, while the WST-1 assay indicated that the non-PEGylated formulation altered mitochondrial activity more significantly compared to the PEGylated ones. The study underlined that the selection of appropriate cytotoxicity assays should be based on the possible mechanism of cellular perturbation. Alternatively, different aspects of cellular toxicity should be tested. Additionally, there is a need for well-designed controls to overcome nanoparticle scattering effects and avoid potentially false high toxicity results, which was demonstrated. Combining orthogonal, well-designed physicochemical and biological assays in a standardized manner as an initial step in the reliable preclinical characterization of nanomedicines is suggested. This represents a key aspect of new methodologies in nanomedicine characterization.

Mutational interference with oligomerization properties of OCP-related apo- and holoproteins studied by analytical ultracentrifugation.

In this study, the oligomerization pattern of apo- and holoforms of the Orange Carotenoid Protein (OCP) was examined under different conditions such as photoactivation state, concentration, and carotenoid embedment using analytical ultracentrifugation. Furthermore, studies were conducted on OCP constructs carrying point mutations of amino acid residues affecting OCP oligomerization. Our findings reveal that the concentration-dependent dimerization of dark-adapted OCP holoprotein from Synechocystis sp. PCC 6803 can be effectively prevented by the R27L mutation in the OCP-NTD. By introducing the E258R mutation (also in conjunction with R27L) into the OCP-CTD, monomeric OCP apoprotein can be obtained. Additionally, the holoprotein of the dark-adapted OCP-R27L/E258R variant was monomeric, and, supported by size-exclusion chromatography experiments, the photoactivated form of the OCP-R27L/E258R variant was monomeric as well. This variant, which does not oligomerize in either photocycle state, returns from the photoactivated to the dark-adapted state at a significantly faster rate than the OCP wild-type and the R27L mutant thereof. These observations also highlight the crucial interdependence between OCP dimerization in both photocycle states, the lifetime of the photoactive state of OCP, and the kinetics of the OCP photocycle.

Tau destabilization in a familial deletion mutant K280 accelerates its fibrillization and enhances the seeding effect.

Tauopathies cover a range of neurodegenerative diseases in which natively unfolded tau protein aggregates and spreads in the brain during disease progression. To gain insights into the mechanism of tau structure and spreading, here, we examined the biochemical and cellular properties of human full-length wild-type and familial mutant tau, ΔK280, with a deletion at lysine 280. Our results showed that both wild-type and mutant tau are predominantly monomeric by analytical ultracentrifugation. The mutant tau may lose intramolecular contacts and is significantly destabilized assessed by cross-linking mass spectrometry and urea denaturation. Moreover, the mutant tau displayed accelerated fibril formation compared to the wild-type tau. Upon cross-seeding, the wild-type tau was seeded more easily by wild-type seeds than mutant seeds showing that homotypic seeding is more efficient. The wild-type tau was successfully converted to fibrils with mutant signatures by mutant seeds. Live cell cross-correlation fluorescence spectroscopy studies indicated that wild-type tau forms trimeric species and the mutant tau forms a larger assembly and processes higher cell-to-cell transmission. Overall, these findings shed light on the fundamental mechanisms of tau structure/stability, aggregation, and seeding to facilitate future therapeutic development for tauopathies.

Effective Thermal Conductivity of Cyclohexane-Based Nanofluids Containing Cerium Dioxide Nanoparticles with Chemisorbed Organic Shell

In the present study, the effective thermal conductivity λeff of nanofluids containing metal oxide nanoparticles with a chemisorbed organic shell was investigated experimentally and theoretically. The model systems synthesized by a continuous-flow hydrothermal method consist of cyclohexane as organic base fluid and dispersed nearly spherical cerium dioxide (CeO2) core nanoparticles with a decanoic acid shell chemically attached to their surface. From the differences between the hydrodynamic diameters of the two core–shell nanoparticle types with (8.6 or 9.1) nm determined by dynamic light scattering (DLS) and the nearly spherical CeO2 core diameters obtained by analytical ultracentrifugation (AUC) and transmission electron microscopy (TEM), an estimation for the thickness of the entire hydrodynamic layer around the particle core in the range of about (1.1 to 1.3) nm could be deduced. Experimental data for λeff of the nanofluids and the thermal conductivity of the base fluid λbf were determined with a steady-state guarded parallel-plate instrument (GPPI) with an expanded (k = 2) relative uncertainty of 0.026 at atmospheric pressure over a temperature range from (283.15 to 313.15) K in steps of 10 K. The measurement results for the thermal-conductivity ratio λeff ·λbf–1 are independent of temperature and increase with increasing volume fraction of the CeO2 core nanoparticles up to about 0.023. It was found that the experimental results can be described by the Hamilton–Crosser model within their experimental uncertainties for all temperatures investigated.

Interlaboratory Measurement of Adeno-Associated Virus: Comparative Quantification of Full and Empty Capsids.

Recombinant adeno-associated virus (AAV) is one of the main viral vector-based gene therapy platforms. AAV is a virus consisting of a ≈25 nm diameter capsid with a ≈4.7 kb cargo capacity. Therapeutic safety and efficacy depend on the correct encapsidation of the DNA in individual virus particles, which is often characterized by the single scalar value of the ratio of full capsids with complete viral genomes to the total viral capsid number [the full-to-total (FTT) ratio]. This study reports on the interlaboratory and intertechnique variations of measurement methods for FTT among a cohort of organizations. The analytical methods used were sedimentation velocity analytical ultracentrifugation (SV-AUC) with UV/Vis and/or Rayleigh interference optics, size exclusion chromatography (SEC) with multi-angle light scattering (MALS), and tandem UV/Vis and/or refractive index, cryogenic electron microscopy, dual-wavelength ultraviolet spectrophotometry, and ELISA coupled with quantitative PCR (qPCR, dPCR, or ddPCR). FTT measurements for both AAV5 and AAV8 serotypes were similar, except for PCR-ELISA. The optical techniques (UV spectroscopy/SEC-MALS) showed <10% SD between laboratories, likely from the uniformity of existing industry protocols. AUC, while demonstrating good repeatability, had ≈25% SD interlaboratory, suggesting the need for standardized methods. PCR and ELISA had poor reproducibility due to variations in both PCR and ELISA protocols and instrumentation. The discussion presents intended future efforts to improve and harmonize these measurements to increase both the repeatability and reproducibility of AAV viral particle critical quality attributes such as FTT.

Solution biophysics identifies lipid nanoparticle non-sphericity, polydispersity, and dependence on internal ordering for efficacious mRNA delivery.

Lipid nanoparticles (LNPs) are the most advanced delivery system currently available for RNA therapeutics. Their development has accelerated since the success of Patisiran, the first siRNA-LNP therapeutic, and the mRNA vaccines that emerged during the COVID-19 pandemic. Designing LNPs with specific targeting, high potency, and minimal side effects is crucial for their successful clinical use. These characteristics have been improved through the development of microfluidic platforms, which have enhanced the efficacy and uniformity of LNP batches. However, our understanding of how the composition and mixing method influences the structural, biophysical, and biological properties of the resulting particles remains limited, hindering the development of LNPs. Our lack of structural understanding extends from the physical and compositional polydispersity of LNPs, which render traditional characterization methods, such as dynamic light scattering (DLS), unable to accurately quantitate the physicochemical characteristics of LNPs. In this study, we address the challenge of structurally characterizing polydisperse LNP formulations using emerging solution-based biophysical methods that have higher resolution and provide biophysical data beyond size and polydispersity. These techniques include sedimentation velocity analytical ultracentrifugation (SV-AUC), field flow fractionation followed by multi-angle light scattering (FFF-MALS), and size-exclusion chromatography in-line with synchrotron small-angle X-ray scattering (SEC-SAXS). Here, we show that the LNPs have intrinsic polydispersity in size, RNA loading, and shape, and that these parameters are dependent on both the formulation technique and lipid composition. Lastly, we demonstrate that these biophysical methods can be employed to predict transfection in three biological models by examining the relationship between mRNA translation and physicochemical characteristics. We envision that employing solution-based biophysical methods will be essential for determining LNP structure-function relationships, facilitating the creation of new design rules for future LNPs.

Synthetic T-Cell Receptor-like Protein Behaves as a Janus Particle in Solution.

Protein engineering enables the creation of tailor-made proteins for a variety of applications. ImmTACs stand out as promising therapeutics for cancer and other treatments while also presenting unique challenges for stability, formulation, and delivery. We have shown that ImmTACs behave as Janus particles in solution, leading to self-association at low concentrations, even when the average protein-protein interactions suggest that the molecule should be stable. The formation of small but stable oligomers was confirmed by static and dynamic light scattering and analytical ultracentrifugation. Modeling of the structure using AlphaFold leads to a rational explanation for this behavior, consistent with the Janus particle assembly observed for inverse patchy particles.

Amino acids modulate liquid–liquid phase separation in vitro and in vivo by regulating protein–protein interactions

Liquid-liquid phase separation (LLPS) is an intracellular process widely used by cells for many key biological functions. It occurs in complex and crowded environments, where amino acids (AAs) are vital components. We have found that AAs render the net interaction between proteins more repulsive. Here, we find that some AAs efficiently suppress LLPS in test tubes (in vitro). We then screen all the proteinogenic AAs and find that three specific AAs, including proline, glutamine, and glycine, significantly suppressed the formation of stress granules (SGs) in U2OS and HeLa cell lines (in vivo) irrespective of stress types. We also observe the effect in primary fibroblast cells, a viable cell model for neurodegenerative disorders. Kinetic studies by live-cell microscopy show that the presence of AAs not only slows down the formation but also decreases the saturating number and prevents the coalescence of SGs. We finally use sedimentation-diffusion equilibrium analytical ultracentrifuge (SE-AUC) to demonstrate that the suppression effects of AAs on LLPS may be due to their modulation in protein-protein and RNA-RNA interactions. Overall, this study reveals an underappreciated role of cellular AAs, which may find biomedical applications, especially in treating SG-associated diseases.

Assembly landscape of the complete B-repeat superdomain from Staphylococcus epidermidis strain 1457.

The accumulation-associated protein (Aap) is the primary determinant of Staphylococcus epidermidis device-related infections. The B-repeat superdomain is responsible for intercellular adhesion that leads to the development of biofilms occurring in such infections. It was recently demonstrated that Zn-induced B-repeat assembly leads to formation of functional amyloid fibrils, which offer strength and stability to the biofilm. Rigorous biophysical studies of Aap B-repeats from S. epidermidis strain RP62A revealed Zn-induced assembly into stable, reversible dimers and tetramers, prior to aggregation into amyloid fibrils. Genetic manipulation is not tractable for many S. epidermidis strains, including RP62A; instead, many genetic studies have used strain 1457. Therefore, to better connect findings from biophysical and structural studies of B-repeats to in vivo studies, the B-repeat superdomain from strain 1457 was examined. Differences between the B-repeats from strain RP62A and 1457 include the number of B-repeats, which has been shown to play a critical role in assembly into amyloid fibrils, as well as the distribution of consensus and variant B-repeat subtypes, which differ in assembly competency and thermal stability. Detailed investigation of the Zn-induced assembly of the full B-repeat supderdomain from strain 1457 was conducted using analytical ultracentrifugation. Whereas the previous construct from RP62A (Brpt5.5) formed a stable tetramer prior to aggregation, Brpt6.5 from 1457 forms extremely large stable species on the order of ∼28-mers, prior to aggregation into similar amyloid fibrils. Our data suggest that both assembly pathways may proceed through the same mechanism of dimerization and tetramerization, and both conclude with the formation of amyloid-like fibrils. Discussion of assembly behavior of B-repeats from different strains and of different length is provided with considerations of biological implications.

Studying C9orf72 dipeptide repeat polypeptide aggregation using an analytical ultracentrifuge equipped with fluorescence detection

Sedimentation velocity, using an analytical ultracentrifuge equipped with fluorescence detection, and electrophoresis methods are used to study aggregation of proteins in transgenic animal model systems. Our previous work validated the power of this approach in an analysis of mutant huntingtin aggregation. We demonstrate that this method can be applied to another neurodegenerative disease studying the aggregation of three dipeptide repeats (DPRs) produced by aberrant translation of mutant c9orf72 containing large G4C2 hexanucleotide repeats. These repeat expansions are the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We analyzed the aggregation patterns of (Gly-Pro)47, (Gly-Ala)50, and (Gly-Arg)50 fused to fluorescent proteins in samples prepared from D. melanogaster, and (Gly-Ala)50 in C. elegans, using AU-FDS and SDD-AGE. Results suggest that (GP)47 is largely monomeric. In contrast, (GA)50 forms both intermediate and large-scale aggregates. (GR)50 is partially monomeric with some aggregation noted in SDD-AGE analysis. The aggregation of this DPR is likely to represent co-aggregated states with DNA and/or RNA. The power of these methods is the ability to gather data on aggregation patterns and characteristics in animal model systems, which may then be used to interpret the mitigation of aggregation through genetic or molecular therapeutic interventions.

Impact of primary sequence changes on the self-association properties of mammalian cystathionine beta-synthase enzymes.

Cystathionine beta-synthase (CBS) is an evolutionarily conserved enzyme that plays a key role in mammalian sulfur amino acid biochemistry, mutations in which are the cause of classical homocystinuria (HCU), an inborn error of metabolism. Although there is agreement in the literature that CBS is a homomultimer, its precise structure is a source of confusion. Here, we performed a series of experiments examining the quaternary structure of various wild-type and mutant CBS enzymes using a combination of native gel electrophoresis, in situ activity assays, analytical ultracentrifugation, and gel filtration. Our data show that recombinantly expressed and purified full-length wild-type human CBS enzyme (hCBS) and HCU-causing variants (p.P422L, p.I435T, and p.R125Q CBS) form high molecular weight assemblies that are consistent with the properties expected of a filament. The filament is enzymatically active, and its size is sensitive to protein concentration. This behavior contrasts sharply with hCBS enzymes containing small deletions within the Bateman domain, which form stable tetramers and octamers regardless of concentration. Examination of liver lysates from humans and mice confirms the existence of enzymatically active high molecular weight aggregates in vivo, but also shows that these aggregates are specific to human CBS and do not occur in mice. Molecular modeling using AlphaFold2 suggests that these experimentally observed differences may be explained by subtle differences in the interaction mediated by the Bateman domains. Our results show that small differences in amino acid sequence can cause large differences in the size and shape of CBS multimers.

From Ions to Crystals: A Comprehensive View of the Non-Classical Nucleation of Calcium Sulfate.

The early stages of mineralization continue to be in the focus of intensive research due to their inherent importance for natural and engineered environments. While numerous observations have been reported for single steps in the pathways of various crystallizing phases in previous studies, the complexity of the underlying processes and their elusive character have left central questions unanswered in most cases. In the present work, we provide a detailed view on the nucleation of calcium sulfate mineralization-an abundant mineral with broad use in construction industry-in aqueous systems at ambient conditions. As experimental basis, a co-titration procedure with potentiometric, turbidimetric and conductometric detection was developed, allowing solution speciation and the formation of crystallization precursors to be monitored quantitatively as the level of nominal (super)saturation gradually increases. The nature and spatiotemporal evolution of these precursors was further elucidated by time-resolved small-angle X-ray scattering (SAXS) and analytical ultracentrifugation (AUC) experiments, complemented by cryogenic transmission electron microscopy (cryo-TEM) as a direct imaging technique. The results reveal how ions associate into nanometric primary species, which subsequently aggregate and develop anisotropic order by intrinsic structural reorganization. Our observations challenge the common understanding of fundamental notions such as the nucleation barrier or the meaning of supersaturation, with broad implications for mineralization phenomena in general and the formation of calcium sulfate in geochemical settings and industrial applications in particular.

Von Ionen zu Kristallen: Neue Einblicke die nicht‐klassische Keimbildung von Calciumsulfat

AbstractDie frühen Stadien der Mineralisierung stehen aufgrund ihrer inhärenten Bedeutung für Prozesse in natürlicher Umgebung sowie in technischen Anwendungen weiterhin im Fokus intensiver Forschung. Obwohl bereits viele wertvolle Beobachtungen für einzelne Schritte in den verschiedenen Phasen eines Kristallisationsvorgangs berichtet wurden, blieben wesentliche Fragen zumeist unbeantwortet aufgrund der Komplexität der zugrundeliegenden Mechanismen und des transienten Charakters der beteiligten Vor‐ und Zwischenstufen. In der vorliegenden Arbeit geben wir einen detaillierten Überblick zur Keimbildung von Calciumsulfat (einem Mineral mit großer Bedeutung für die Bauindustrie) in wässrigen Systemen bei Umgebungsbedingungen. Als experimentelle Grundlage wurde ein Titrationsverfahren mit gleichzeitiger potentiometrischer, turbidimetrischer und konduktometrischer Detektion entwickelt, um die Assoziation von Ionen in Lösung und die Bildung von Vorläuferstrukturen bei kontinuierlich ansteigender nomineller Übersättigung quantitativ verfolgen zu können. Die Eigenschaften dieser Präkursoren und ihre zeitliche Entwicklung vor und während der Nukleation wurden in situ mittels Kleinwinkel‐Röntgenstreuung (SAXS) und analytischer Ultrazentrifugation (AUC) aufgeklärt. Zusätzlich wurde Transmissionselektronenmikroskopie unter kryogenen Bedingungen (Kryo‐TEM) als bildgebendes Verfahren genutzt. Die Ergebnisse zeigen, wie sich Ionen zu nanometrischen Primärspezies verbinden, die anschließend aggregieren und durch intrinsische strukturelle Reorganisation eine anisotrope Ordnung entwickeln. Unsere Beobachtungen stellen das gängige Bild von grundlegenden Begriffen wie der Keimbildungsbarriere oder der Bedeutung der Übersättigung in Frage. Daraus ergeben sich weitreichende Auswirkungen auf unser Verständnis von Kristallisationsphänomenen im Allgemeinen und der Bildung von Calciumsulfat in geochemischen und angewandten Systemen im Besonderen.

Analysis of Macromolecular Size Distributions in Concentrated Solutions

AbstractThe solution state of macromolecules in concentrated solutions impacts fields ranging from cell biology, to colloid chemistry and engineering of protein pharmaceuticals. Dependent on the interplay between repulsive and weakly attractive forces, proteins may exhibit oligomerization, aggregation, crystallization, liquid‐liquid phase separation, or the formation of multiprotein complexes. The particle size‐distribution is a key characteristic, but difficult to determine when interparticle distances are on the order of their size and macromolecular motion is coupled through hydrodynamic interactions. Here we extend sedimentation velocity analytical ultracentrifugation to measure macromolecular size distributions under these conditions: We apply results from statistical fluid mechanics for the concentration‐dependence of hindered settling and diffusion, embedded in a mean‐field approximation that can resolve coupled sedimentation and diffusion processes of different sized species given experimental sedimentation data. This is combined with a description of transient optical aberrations from lensing in the refractive index gradients associated with sedimentation boundaries (Wiener skewing). We demonstrate this approach in the application to protein solutions with macromolecular volume fractions up to ≈10 %, for example, resolving monomers and dimers of albumin at 140 mg/ml. This enables size‐distribution analysis of proteins at concentrations of therapeutic antibody formations and close to physiological concentration in serum and cells.

Biophysical Analysis of Vip3Aa Toxin Mutants Before and After Activation.

Cry toxins from Bacillus thuringiensis are effective biopesticides that kill lepidopteran pests, replacing chemical pesticides that indiscriminately attack both target and non-target organisms. However, resistance in susceptible pests is an emerging problem. B. thuringiensis also produces vegetative insecticidal protein (Vip3A), which can kill insect targets in the same group as Cry toxins but using different host receptors, making the combined application of Cry and Vip3A an exciting possibility. Vip3A toxicity requires the formation of a homotetramer. Hence, screening of Vip3A mutants for increased stability requires orthogonal biophysical assays that can test both tetrameric integrity and monomeric robustness. For this purpose, we have used herein for the first time a combination of analytical ultracentrifugation (AUC), mass photometry (MP), differential static light scattering (DSLS) and differential scanning fluorimetry (DSF) to test five mutants at domains I and II. Although all mutants appeared more stable than the wild type (WT) in DSLS, mutants that showed more dissociation into dimers in MP and AUC experiments also showed earlier thermal unfolding by DSF at domains IV-V. All of the mutants were less toxic than the WT, but toxicity was highest for domain II mutations N242C and F229Y. Activation of the protoxin was complete and resulted in a form with a lower sedimentation coefficient. Future high-resolution structural data may lead to a deeper understanding of the increased stability that will help with rational design while retaining native toxicity.

Mechanisms of amphibian arrestin 1 self-association and dynamic distribution in retinal photoreceptors

Visual arrestin 1 (Arr1) is an essential protein for termination of the light response in photoreceptors. While mammalian Arr1s form dimers and tetramers at physiological concentrations in vitro, oligomerization in other vertebrates has not been studied. Here we examine self-association of Arr1 from two amphibian species, Xenopus laevis (xArr1), and Ambystoma tigrinum (salArr1). Sedimentation velocity analytical ultracentrifugation showed that xArr1 and salArr1 oligomerization is limited to dimers. The KD for dimer formation was 53 μM for xArr1 and 44 μM for salArr1, similar to the 69 μM KD for bovine Arr1 (bArr1) dimers. Mutations of orthologous amino acids important for mammalian Arr1 oligomerization had no impact on xArr1 dimerization. Crystallography showed that the fold of xArr1 closely resembles that of bArr1 and crystal structures in different space groups revealed two potential xArr1 dimer forms: a symmetrical dimer with a C-domain interface (CC dimer), resembling the bArr1 solution dimer, and an asymmetric dimer with an N-domain/C-domain interface. Mutagenesis of residues predicted to interact in either of these two dimer forms yielded modest reduction in dimer affinity, suggesting that the dimer interfaces compete or are not unique. Indeed, small-angle X-ray scattering and protein painting data were consistent with a symmetric anti-parallel solution dimer (AP dimer) distinct from the assemblies observed by crystallography. Finally, a computational model evaluating xArr1 binding to compartment-specific partners and partitioning based on heterogeneity of available cytoplasmic spaces shows that Arr1 distribution in dark adapted photoreceptors is largely explained by the excluded volume effect together with tuning by oligomerization.