Impact of Glycosylation on Protein-Protein Self-Interactions of Monoclonal Antibodies.

Protein self-interactions measured via second osmotic virial coefficients (B22) and dynamic light scattering interaction parameter values (kD) are often used as metrics for assessing the favorability of protein candidates and different formulations during monoclonal antibody (MAb) product development. Model predictions of B22 or kD typically do not account for glycans, though glycosylation can potentially impact experimental MAb self-interactions. To the best of our knowledge, the impact of MAb glycosylation on the experimentally measured B22 and kD values has not yet been reported. B22 and kD values of two fully deglycosylated MAbs and their native (i.e., fully glycosylated) counterparts were measured by light scattering over a range of pH and ionic strength conditions. Significant differences between B22 and kD of the native and deglycosylated forms were observed at a range of low to high ionic strengths used to modulate the effect of electrostatic contributions. Differences were most pronounced at low ionic strength, indicating that electrostatic interactions are a contributing factor. Though B22 and kD values were statistically equivalent at high ionic strengths where electrostatics were fully screened, we observed protein-dependent qualitative differences, which indicate that steric interactions may also play a role in the observed B22 and kD differences. A domain-level coarse-grained molecular model accounting for charge differences was considered to potentially provide additional insight but was not fully predictive of the behavior across all of the solution conditions investigated. This highlights that both the level of modeling and lack of inclusion of glycans may limit existing models in making quantitatively accurate predictions of self-interactions.

Experimental support for reclassification of the light scattering second virial coefficient from macromolecular solutions as a hydrodynamic parameter.

This investigation examines the source of the disparity between experimental values of the light scattering second virial coefficient [Formula: see text] (mL.mol/g2) for proteins and those predicted on the statistical mechanical basis of excluded volume. A much better theoretical description of published results for lysozyme is obtained by considering the experimental parameters to monitor the difference between the thermodynamic excluded volume term and its hydrodynamic counterpart. This involves a combination of parameters quantifying concentration dependence of the translational diffusion coefficient obtained from dynamic light scattering measurements. That finding is shown to account for observations of a strong correlation between [Formula: see text] (mL/g), where M2 is the molar mass (molecular weight) of the macromolecule and the diffusion concentration parameter [Formula: see text] (mL/g). On the grounds that [Formula: see text] is regarded as a hydrodynamic parameter, the same status should be accorded the light scattering second virial coefficient rather than its current incorrect thermodynamic designation as [Formula: see text] (mL.mol/g2), or just B, the osmotic second virial coefficient for protein self-interaction.

In situ characterization of the agglutination of lectins via cross-linking of carbohydrates by time-resolved measurement of forward static light scattering.

We present real-time observations of a structurally variable process for cross-linking agglutination between multivalent lectins and glycoclusters using a small-angle forward static light scattering (F-SLS) technique. In this study, a cross-linking agglutination reaction was carried out using a tetravalent Neu5Acα2,6LacNAc-glycocluster and Sambucus sieboldiana agglutinin (SSA). The scattering intensity of time-resolved F-SLS increased with formation of the Neu5Acα2,6LacNAc-glycocluster-SSA cross-linked complex. Using this approach, fine sequential cross-linking agglutination between glycoclusters and lectins was observed in real-time. The rate of increase in the intensity of time-resolved F-SLS increased with the concentration of sialo-glycoclusters and SSA. Structural analysis based on the fractal dimension using time-resolved F-SLS patterns revealed that the density of the aggregates changed with progression of the cross-linking reaction until equilibrium was reached. This is the first report to evaluate the cross-linking agglutination reaction between glycoclusters and lectins and analysis of the subsequent structure of the obtained aggregates using time-resolved measurements of F-SLS.

Self-assembly of smooth muscle myosin filaments: adaptation of filament length by telokin and Mg·ATP.

The contractile apparatus of smooth muscle is malleable to accommodate stress and strain exerted on the muscle cell and to maintain optimal contractility. Structural lability of smooth muscle myosin filaments is believed to play an important role in the cell's malleability. However, the mechanism and regulation of myosin filament formation is still poorly understood. In the present in vitro study, using a static light scattering method, length distributions were obtained from suspensions of short myosin filaments (SFs) formed by rapid dilution or long ones (LFs) formed by slow dialysis. The distributions indicated the presence of dynamic equilibriums between soluble myosin and the SFs; i.e.: trimers, hexamers and mini filaments, covering the range up to 0.75 µm. The LFs were more stable, exhibiting favorable sizes at about 1.25, 2.4 and 4.5 µm. More distinct distributions were obtained from filaments adsorbed to a glass surface, by evanescent wave scattering and local electric field enhancement. Addition of telokin (TL) to the suspensions of unphosphorylated SFs resulted in widening of the soluble range, while in the case of the LFs this shift was larger, and accompanied by reduced contribution of the soluble myosin species. Such changes were largely absent in the case of phosphorylated myosin. In contrast, the presence of Mg·ATP resulted in elongation of the filaments and clear separation of filaments from soluble myosin species. Thus, TL and Mg·ATP appeared to modify the distribution of myosin filament lengths, i.e., increasing the lengths in preparing for phosphorylation, or reducing it to aid dephosphorylation.

Toward Huygens' Sources with Dodecahedral Plasmonic Clusters.

The design and chemical synthesis of plasmonic nanoresonators exhibiting a strong magnetic response in the visible is a key requirement to the realization of efficient functional and self-assembled metamaterials. However, novel applications like Huygens' metasurfaces or mu-near-zero materials require stronger magnetic responses than those currently reported. Our numerical simulations demonstrate that the specific dodecahedral morphology, whereby 12 silver satellites are located on the faces of a nanosized dielectric dodecahedron, provides sufficiently large electric and magnetic dipolar and quadrupolar responses that interfere to produce so-called generalized Huygens' sources, fulfilling the generalized Kerker condition. Using a multistep colloidal engineering approach, we synthesize highly symmetric plasmonic nanoclusters with a controlled silver satellite size and show that they exhibit a strong forward scattering that may be used in various applications such as metasurfaces or perfect absorbers.

Application of Dynamic and Static Light Scattering for Size and Shape Characterization of Small Extracellular Nanoparticles in Plasma and Ascites of Ovarian Cancer Patients.

In parallel to medical treatment of ovarian cancer, methods for the early detection of cancer tumors are being sought. In this contribution, the use of non-invasive static (SLS) and dynamic light scattering (DLS) for the characterization of extracellular nanoparticles (ENPs) in body fluids of advanced serous ovarian cancer (OC) and benign gynecological pathology (BP) patients is demonstrated and critically evaluated. Samples of plasma and ascites (OC patients) or plasma, peritoneal fluid, and peritoneal washing (BP patients) were analyzed. The hydrodynamic radius (Rh) and the radius of gyration (Rg) of ENPs were calculated from the angular dependency of LS intensity for two ENP subpopulations. Rh and Rg of the predominant ENP population of OC patients were in the range 20-30 nm (diameter 40-60 nm). In thawed samples, larger particles (Rh mostly above 100 nm) were detected as well. The shape parameter ρ of both particle populations was around 1, which is typical for spherical particles with mass concentrated on the rim, as in vesicles. The Rh and Rg of ENPs in BP patients were larger than in OC patients, with ρ ≈ 1.1-2, implying a more elongated/distorted shape. These results show that SLS and DLS are promising methods for the analysis of morphological features of ENPs and have the potential to discriminate between OC and BP patients. However, further development of the methodology is required.

Solution structure and flexibility of the condensin HEAT-repeat subunit Ycg1.

High-resolution structural analysis of flexible proteins is frequently challenging and requires the synergistic application of different experimental techniques. For these proteins, small-angle X-ray scattering (SAXS) allows for a quantitative assessment and modeling of potentially flexible and heterogeneous structural states. Here, we report SAXS characterization of the condensin HEAT-repeat subunit Ycg1Cnd3 in solution, complementing currently available high-resolution crystallographic models. We show that the free Ycg1 subunit is flexible in solution but becomes considerably more rigid when bound to its kleisin-binding partner protein Brn1Cnd2 The analysis of SAXS and dynamic and static multiangle light scattering data furthermore reveals that Ycg1 tends to oligomerize with increasing concentrations in the absence of Brn1. Based on these data, we present a model of the free Ycg1 protein constructed by normal mode analysis, as well as tentative models of Ycg1 dimers and tetramers. These models enable visualization of the conformational transitions that Ycg1 has to undergo to adopt a closed rigid shape and thereby create a DNA-binding surface in the condensin complex.

Temperature Dependence of Protein Solution Viscosity and Protein-Protein Interactions: Insights into the Origins of High-Viscosity Protein Solutions.

Protein solution viscosity (η) as a function of temperature was measured at a series of protein concentrations under a range of formulation conditions for two monoclonal antibodies (MAbs) and a globular protein (aCgn). Based on theoretical arguments, a strong temperature dependence for protein-protein interactions (PPI) indicates highly anisotropic, short-ranged attractions that could lead to higher solution viscosities. The semi-empirical Ross-Minton model was used to determine the apparent intrinsic viscosity, shape, and "crowding" factors for each protein as a function of temperature and formulation conditions. The apparent intrinsic viscosity was independent of temperature for aCgn, while a slight decrease with increasing temperature was observed for the MAbs. The temperature dependence of solution viscosity was analyzed using the Andrade-Eyring equation to determine the effective activation energy of viscous flow (Ea,η). While Ea,η values were different for each protein, they were independent of formulation conditions for a given protein. PPI were quantified via the osmotic second virial coefficient (B22) and the protein diffusion interaction parameter (kD) as a function of temperature under the same formulation conditions as the viscosity measurements. Net interactions ranged from strongly attractive to repulsive by changing formulation pH and ionic strength for each protein. Overall, larger activation energies for PPI corresponded to larger activation energies for η, and those were predictive of the highest η values at higher protein concentrations.

Study of the complex coacervation mechanism between ovalbumin and the strong polyanion PSSNa: influence of temperature and pH.

We studied the complex between ovalbumin and long flexible poly-(sodium 4-styrene sulfonate) as a function of pH and temperature. We used various techniques [turbidimetry, conductometry, dynamic light scattering, viscosimetry, and ultra-small-angle light scattering (USALS)] to fully characterize the coacervate complex. Different phases of complexation versus temperature were determined by turbidimetric analysis (pHc, pHϕ1, and pHϕ2). The optimal protein/polyelectrolyte interaction occurred at pHopt 4. An increase in temperature made the hydrophobic interactions more favorable in the case of the soluble complex and complex coacervation phases (pH > pHϕ2). We systematically determined the activation energy to follow the conformational changes of the complex at different temperatures. At pHopt, the size of the formed complex showed a remarkable decrease with temperature increase. USALS was used to determine simultaneously the radius of gyration (Rg) and fractal dimension Df of the coacervate.

Effects of High-Intensity Ultrasound Pretreatment on Structure, Properties, and Enzymolysis of Soy Protein Isolate.

The objective of this study was to investigate the effects of different high-intensity ultrasonication (HIU) pretreatment on the structure and properties of soybean protein isolate (SPI) as well as enzymatic hydrolysis of SPI by bromelain and antioxidant activity of hydrolysates. The HIU-treated SPI fractions showed a decrease in the proportion of α-helices and ß-turns and an increase in the content of ß-sheets and random coils based on Fourier-transform infrared spectroscopy. Near-infrared spectra and fluorescence spectra analyses provided support for the changes in secondary and tertiary structures of SPI after ultrasound treatment. The particle size of SPI decreased from 217.20 nm to 141.23 nm and the absolute zeta potential increased. Scanning electron microscopy showed that HIU treatment changed apparent morphology. Dynamic and static light scattering of ultrasonicated samples showed that SPI structure had changed from hard-sphere to hollow-sphere or polydisperse and monodisperse gaussian coils. HIU pretreatment significantly increased the hydroxyl-radical scavenging and the degree of hydrolysis of the SPI hydrolysates.

Characterising protein/detergent complexes by triple-detection size-exclusion chromatography.

BACKGROUND: In vitro investigations of membrane proteins usually depend on detergents for protein solubilisation and stabilisation. The amount of detergent bound to a membrane protein is relevant to successful experiment design and data analysis but is often unknown. Triple-detection size-exclusion chromatography enables simultaneous separation of protein/detergent complexes and protein-free detergent micelles and determination of their molar masses in a straightforward and absolute manner. Size-exclusion chromatography is used to separate different species, while ultraviolet absorbance, static light scattering, and refractive index measurements allow molar mass determination of protein and detergent components. RESULTS: We refined standard experimental and data-analysis procedures for challenging membrane-protein samples that elude routine approaches. The general procedures including preparatory steps, measurements, and data analysis for the characterisation of both routine and complex samples in difficult solvents such as concentrated denaturant solutions are demonstrated. The applicability of the protocol but also its limitations and possible solutions are discussed, and an extensive troubleshooting section is provided. CONCLUSIONS: We established and validated a protocol for triple-detection size-exclusion chromatography that enables the inexperienced user to perform and analyse measurements of well-behaved protein/detergent complexes. More experienced users are provided with an example of a more sophisticated analysis procedure allowing mass determination under challenging separation conditions.

Physical characterization and profiling of airway epithelial derived exosomes using light scattering.

Exosomes and other extracellular vesicles have been gaining interest during the last decade due to their emerging role in biology and, disease pathogenesis and their biomarker potential. Almost all published research related to exosomes and other extracellular vesicles include some form of physical characterization. Therefore, these vesicles should be precisely profiled and characterized physically before studying their biological role as intercellular messengers, biomarkers or therapeutic tools. Using a combination of light scattering techniques, including dynamic light scattering (DLS) and multi-angle laser light scattering combined with size exclusion separation (SEC-MALLS), we physically characterized and compared distinct extracellular vesicles derived from the apical secretions of two different cultured airway epithelial cells. The results indicated that epithelial cells release vesicles with distinct physical properties and sizes. Human primary tracheobronchial cell culture (HTBE) derived vesicles have a hydrodynamic radius (Rh) of approximately 340 nm while their radius of gyration (Rg) is approximately 200 nm. Electron microscopy analysis, however, revealed that their spherical component is 40-100 nm in size, and they carry filamentous, entangled membrane mucins on their surface that increases their overall radius. The mucin decoration on the surface defines their size and charge as measured using light scattering techniques. Their surface properties mirror the properties of the cells from which they are derived. This may provide a unique tool for researchers to elucidate the unanswered questions in normal airway biology and innate and adaptive defense, including the remodeling of airways during inflammation, tumorigenesis and metastasis.

A method to Quantify the Affinity of Cabazitaxel for PLA-PEG Nanoparticles and Investigate the Influence of the Nano-Assembly Structure on the Drug/Particle Association.

PURPOSE: To study the impact of the size and the structure of the nano-assembly on the drug/particle association, determining the intrinsic partition coefficient, in order to better master the encapsulation and release properties of the carrier. METHODS: An experimental methodology is proposed to characterize the drug/nanoparticle association by mean of a partition coefficient between the PLA-PEG nanoparticles and the suspending aqueous medium, referred to as Kp. The determination was made from apparent values (referred to as Kp (ap)) measured in the presence of solubilizing agents (albumin and hydroxypropyl-ßcyclodextrin) and extrapolation to zero concentration. The structure of nanoparticles was investigated by Transmission Electron Microscopy and static light scattering. RESULTS: Depending on the manufacturing process and the PEG length of the copolymer, the nanoparticles structured either as aggregates of copolymer chains or micelles exhibiting significantly different Kp values. CONCLUSION: The methodological tool described here showed that the difference in cabazitaxel/nanoparticle association between aggregates and micelles could be attributed to the difference in PLA-PEG chains packing.

Heparin induced dimerization of APP is primarily mediated by E1 and regulated by its acidic domain.

The amyloid precursor protein (APP) and its cellular processing are believed to be centrally involved in the etiology of Alzheimer's disease (AD). In addition, many physiological functions have been described for APP, including a role in cell-cell- and cell-ECM-adhesion as well as in axonal outgrowth. We show here the molecular determinants of the oligomerization/dimerization of APP, which is central for its cellular (mis)function. Using size exclusion chromatography (SEC), dynamic light scattering and SEC-coupled static light scattering we demonstrate that the dimerization of APP is energetically induced by a heparin mediated dimerization of the E1 domain, which results in a dimeric interaction of E2. We also show that the acidic domain (AcD) interferes with the dimerization of E1 and propose a model where both, cis- and trans-dimerization occur dependent on cellular localization and function.

Development and validation of high-performance size exclusion chromatography methods to determine molecular size parameters of Haemophilus influenzae type b polysaccharides and conjugates.

Current vaccines against Haemophilus influenzae type b (Hib) consist of the polyribosyl ribitol phosphate (PRP) capsular polysaccharide chemically conjugated to a carrier protein. Among the various biological and physical analyses to be performed on these vaccines, the determination of the molecular size of the polysaccharide preparations throughout the conjugation process is particularly relevant. Comparison of results from high-performance size exclusion chromatography (HPSEC) with those routinely obtained using conventional gel permeation chromatography (CGPC) methods highlights the correlation between the two methods for determining the values of the chromatographic distribution coefficient (KD) of native and activated polysaccharides. The resulting data showed that the KD value is sufficient to characterize these polysaccharides using an HPSEC method. However, additional molecular size parameters (i.e., molar mass and hydrodynamic radius) are necessary for a reliable characterization of the tetanus conjugate (PRP-T), certainly due to the lattice-like structure of the conjugate. In practice, an absolute detection system in HPSEC composed of a low-angle light scattering detector, a viscometer, and a refractive index (RI) detector was used. As demonstrated, these HPSEC methods are rapid, accurate, and reproducible for the polysaccharides and their glycoconjugates and provide a relevant and more informative alternative to the current CGPC methods.

On-line coupling of hollow-fiber flow field-flow fractionation and depolarized multi-angle static light scattering (HF5/D-MALS). Proof of principle.

Introduced here is the on-line coupling of hollow-fiber flow field-flow fractionation (HF5) to depolarized multi-angle static light scattering (D-MALS). HF5 is a size-based separation alternative to size-exclusion and hydrodynamic chromatography and asymmetric flow field-flow fractionation. HF5 can separate larger sizes than its chromatographic counterparts and provides several advantages over its fractionation counterpart, including reduced sample consumption and greater ease of operation. D-MALS is a variant of MALS in which the depolarized scattering from the analyte solution is measured at a variety of angles simultaneously. Measurements of depolarized scattering have previously been employed in studying the optical properties of solutions or suspensions, to determine the length of rod-like analytes, and to gain increased accuracy in the determination of analyte molar mass. The coupling HF5/D-MALS allows for the depolarization ratio of a solution or suspension to be measured continuously across the fractogram. This is demonstrated here for a Teflon latex the size range of which extends beyond that accessible to commercial size-exclusion columns. The results presented provide the first reported on-line HF5/D-MALS coupling, showing the feasibility of the technique as well as its realized potential for providing continuous depolarization measurements, inter alia.

Protein-Mineral Composite Particles with Logarithmic Dependence of Anticancer Cytotoxicity on Concentration of Montmorillonite Nanoplates with Adsorbed Cytochrome c.

Montmorillonite (MM) colloid nanoplates have high adsorption capacity due to their large size/thickness ratio, which allows them to be used as carriers for drug delivery. Upon adsorption of the mitochondrial protein cytochrome c (cytC) onto MM plates, the composite cytC-MM particles acquire anticancer properties because of the ability of cancer cells to phagocytize submicron particles (in contrast to the normal cells). In this way, exogenous cytC can be introduced into tumor cells, thereby triggering apoptosis-an irreversible cascade of biochemical reactions leading to cell death. In the present study, we investigated the physicochemical properties of cytC-MM particles as a function of the cytC concentration in the suspension, namely, the electrophoretic mobility, the mass increment of MM monoplates upon cytC adsorption, the ratio of the adsorbed to the free cytC in the bulk, the protein density on the MM's surface, the number of cytC globules adsorbed on an MM monoplate, the concentration of cytC-MM composite particles in the suspension, and the dependence of cytotoxicity on the cytC-MM particle concentration. For this purpose, we used microelectrophoresis, static and electric light scattering, and a colon cancer cell culture to test the cytotoxic effects of the cytC-MM suspensions. The results show that the cytotoxicity depends linearly on the logarithm of the particle concentration in the cytC-MM suspension reaching 97%.

Light Scattering and Turbidimetry Techniques for the Characterization of Nanoparticles and Nanostructured Networks.

Light scattering and turbidimetry techniques are classical tools for characterizing the dynamics and structure of single nanoparticles or nanostructured networks. They work by analyzing, as a function of time (Dynamic Light Scattering, DLS) or angles (Static Light Scattering, SLS), the light scattered by a sample, or measuring, as a function of the wavelength, the intensity scattered over the entire solid angle when the sample is illuminated with white light (Multi Wavelength Turbidimetry, MWT). Light scattering methods probe different length scales, in the ranges of ~5−500 nm (DLS), or ~0.1−5 µm (Wide Angle SLS), or ~1−100 µm (Low Angle SLS), and some of them can be operated in a time-resolved mode, with the possibility of characterizing not only stationary, but also aggregating, polymerizing, or self-assembling samples. Thus, the combined use of these techniques represents a powerful approach for studying systems characterized by very different length scales. In this work, we will review some typical applications of these methods, ranging from the field of colloidal fractal aggregation to the polymerization of biologic networks made of randomly entangled nanosized fibers. We will also discuss the opportunity of combining together different scattering techniques, emphasizing the advantages of a global analysis with respect to single-methods data processing.

Ultra-/Small Angle X-ray Scattering (USAXS/SAXS) and Static Light Scattering (SLS) Modeling as a Tool to Determine Structural Changes and Effect on Growth in S. epidermidis.

Usually, to characterize bacterial cells' susceptibility to antimicrobials, basic microbiology techniques such as serial dilutions or disk assays are used. In this work, we present an approach focused on combining static light scattering (SLS) and ultra-/small angle X-ray scattering (USAXS/SAXS). This approach was used to support microbiology techniques, with the aim of understanding the structural changes caused to bacteria when they are exposed to different stresses like pH, oxidation, and surfactants. Using USAXS/SAXS and SLS data, we developed a detailed multiscale model for a Gram-positive bacterium, S. epidermidis, and we extracted information regarding changes in the overall size and cell thickness induced by different stresses (i.e., pH and hydrogen peroxide). Increasing the concentration of hydrogen peroxide leads to a progressive reduction in cell wall thickness. Moreover, the concomitant use of pH and hydrogen peroxide provides evidence for a synergy in inhibiting the S. epidermidis growth. These promising results will be used as a starting base to further investigate more complex formulations and improve/refine the data modeling of bacteria in the small angle scattering regime.

Synthesis of Magneto-Controllable Polymer Nanocarrier Based on Poly(N-isopropylacrylamide-co-acrylic Acid) for Doxorubicin Immobilization.

In this work, the preparation procedure and properties of anionic magnetic microgels loaded with antitumor drug doxorubicin are described. The functional microgels were produced via the in situ formation of iron nanoparticles in an aqueous dispersion of polymer microgels based on poly(N-isopropylacrylamide-co-acrylic acid) (PNIPAM-PAA). The composition and morphology of the resulting composite microgels were studied by means of X-ray diffraction, Mössbauer spectroscopy, IR spectroscopy, scanning electron microscopy, atomic-force microscopy, laser microelectrophoresis, and static and dynamic light scattering. The forming nanoparticles were found to be ß-FeO(OH). In physiological pH and ionic strength, the obtained composite microgels were shown to possess high colloid stability. The average size of the composites was 200 nm, while the zeta-potential was -27.5 mV. An optical tweezers study has demonstrated the possibility of manipulation with microgel using external magnetic fields. Loading of the composite microgel with doxorubicin did not lead to any change in particle size and colloidal stability. Magnetic-driven interaction of the drug-loaded microgel with model cell membranes was demonstrated by fluorescence microscopy. The described magnetic microgels demonstrate the potential for the controlled delivery of biologically active substances.