Formation Of Higher-order Aggregates Research Articles

BSA Binding and Aggregate Formation of a Synthetic Amino Acid with Potential for Promoting Fibroblast Proliferation: An In Silico, CD Spectroscopic, DLS, and Cellular Study.

This study presents the chemical synthesis, purification, and characterization of a novel non-natural synthetic amino acid. The compound was synthesized in solution, purified, and characterized using NMR spectroscopy, polarimetry, and melting point determination. Dynamic Light Scattering (DLS) analysis demonstrated its ability to form aggregates with an average size of 391 nm, extending to the low micrometric size range. Furthermore, cellular biological assays revealed its ability to enhance fibroblast cell growth, highlighting its potential for tissue regenerative applications. Circular dichroism (CD) spectroscopy showed the ability of the synthetic amino acid to bind serum albumins (using bovine serum albumin (BSA) as a model), and CD deconvolution provided insights into the changes in the secondary structures of BSA upon interaction with the amino acid ligand. Additionally, molecular docking using HDOCK software elucidated the most likely binding mode of the ligand inside the BSA structure. We also performed in silico oligomerization of the synthetic compound in order to obtain a model of aggregate to investigate computationally. In more detail, the dimer formation achieved by molecular self-docking showed two distinct poses, corresponding to the lowest and comparable energies, with one pose exhibiting a quasi-coplanar arrangement characterized by a close alignment of two aromatic rings from the synthetic amino acids within the dimer, suggesting the presence of π-π stacking interactions. In contrast, the second pose displayed a non-coplanar configuration, with the aromatic rings oriented in a staggered arrangement, indicating distinct modes of interaction. Both poses were further utilized in the self-docking procedure. Notably, iterative molecular docking of amino acid structures resulted in the formation of higher-order aggregates, with a model of a 512-mer aggregate obtained through self-docking procedures. This model of aggregate presented a cavity capable of hosting therapeutic cargoes and biomolecules, rendering it a potential scaffold for cell adhesion and growth in tissue regenerative applications. Overall, our findings highlight the potential of this synthetic amino acid for tissue regenerative therapeutics and provide valuable insights into its molecular interactions and aggregation behavior.

Molecular origin of the two-step mechanism of gellan aggregation

Among hydrocolloids, gellan is one of the most studied polysaccharides due to its ability to form mechanically stable gels. Despite its long-standing use, the gellan aggregation mechanism is still not understood because of the lack of atomistic information. Here, we fill this gap by developing a new gellan force field. Our simulations offer the first microscopic overview of gellan aggregation, detecting the coil to single-helix transition at dilute conditions and the formation of higher-order aggregates at high concentration through a two-step process: first, the formation of double helices and then their assembly into superstructures. For both steps, we also assess the role of monovalent and divalent cations, complementing simulations with rheology and atomic force microscopy experiments and highlighting the leading role of divalent cations. These results pave the way for future use of gellan-based systems in a variety of applications, from food science to art restoration.

The Effect of a Rotating Cone on Horseradish Peroxidase Aggregation on Mica Revealed by Atomic Force Microscopy.

Our study reported herein aims to determine whether an electromagnetic field, induced triboelectrically by a metallic cone, rotating at a frequency of 167 Hz, has an effect on the properties of the horseradish peroxidase (HRP) enzyme. Atomic force microscopy (AFM) was employed to detect even the most subtle effects on single enzyme molecules. In parallel, a macroscopic method (spectrophotometry) was used to reveal whether the enzymatic activity of HRP in solution was affected. An aqueous solution of the enzyme was incubated at a distance of 2 cm from the rotating cone. The experiments were performed at various incubation times. The control experiments were performed with a non-rotating cone. The incubation of the HRP solution was found to cause the disaggregation of the enzyme. At longer incubation times, this disaggregation was found to be accompanied by the formation of higher-order aggregates; however, no change in the HRP enzymatic activity was observed. The results of our experiments could be of interest in the development of enzyme-based biosensors with rotating elements such as stirrers. Additionally, the results obtained herein are important for the correct interpretation of data obtained with such biosensors.

Kinetic analysis of the multistep aggregation pathway of human transthyretin

Aggregation of transthyretin (TTR) is the causative agent for TTR cardiomyopathy and polyneuropathy amyloidoses. Aggregation is initiated by dissociation of the TTR tetramer into a monomeric intermediate, which self-assembles into amyloid. The coupled multiple-step equilibria and low-concentration, aggregation-prone intermediates are challenging to probe using conventional assays. We report a 19F-NMR assay that leverages a highly sensitive trifluoroacetyl probe at a strategic site that gives distinct 19F chemical shifts for the TTR tetramer and monomeric intermediate and enables direct quantification of their populations during the aggregation process. Integration of real-time 19F-NMR and turbidity measurements as a function of temperature allows kinetic and mechanistic dissection of the aggregation pathway of both wild-type and mutant TTR. At physiological temperature, the monomeric intermediate formed by wild-type TTR under mildly acidic conditions rapidly aggregates into species that are invisible to NMR, leading to loss of the NMR signal at the same rate as the turbidity increase. Lower temperature accelerates tetramer dissociation and decelerates monomer tetramerization and oligomerization via reduced hydrophobic interactions associated with packing of a phenylalanine (F87) into a neighboring protomer. As a result, the intermediate accumulates to a higher level, and formation of higher-order aggregates is delayed. Application of this assay to pathogenic (V30M, L55P, and V122I) and protective (T119M) mutants revealed significant differences in behavior. A monomeric intermediate was observed only for V122I: aggregation of V30M and L55P proceeds without an observable monomeric intermediate, whereas the protective mutant T119M remains resistant to tetramer dissociation and aggregation.

What Gives an Insulin Hexamer Its Unique Shape and Stability? Role of Ten Confined Water Molecules.

Self-assembly of proteins often gives rise to interesting quasi-stable structures that serve important biological purposes. Insulin hexamer is such an assembly. While monomer is the biologically active form of insulin, hexamer serves as the storehouse of the hormone. The hexamer also prevents the formation of higher order aggregates. While several studies explored the role of bivalent metal ions like Zn2+, Ca2+, etc., in the stabilization of the hexameric form, the role of water molecules has been ignored. We combine molecular dynamics simulations, quantum calculations, and X-ray analyses to discover that a team of approximately 10 water molecules confined inside a barrel-shaped nanocavity at the center of insulin hexamer is one of the major causes that account for the unusual stability of the biomolecular assembly. These cavity water molecules exhibit interesting dynamical features like intermittent escape and reentrance. We find that these water molecules are dynamically slower than the bulk and weave an intricate hydrogen bond network among themselves and with neighboring protein residues to generate a robust backbone at the center of the hexamer that holds the association strongly from inside and maintains the barrel shape.

Effect of channel-induced shear on biologics during ultrafiltration/diafiltration (UF/DF)

Ultrafiltration/diafiltration (UF/DF) is the standard unit operation in place in biotechnology industry for the final protein concentration and buffer exchange to produce drug substance. Several researchers have observed and published on the formation of higher order aggregates and particulates during UF/DF as a result of recirculation shear and potential exposure to air-liquid interface. This work examines the effect of screen channel-induced shear on particulate formation during UF/DF. To this end, different types of screened channels were examined for processing of monoclonal antibodies and a fusion protein during UF/DF at a fixed crossflow flux. It was found that channel-induced shear significantly affected the product quality for certain biologics, measured using various techniques, such as size-exclusion chromatography (SEC), Micro-Flow Imaging (MFI), dynamic light scattering (DLS), and filterability through a 0.2µm filter. It was demonstrated that cassettes with a more open screened-channel or “lightly suspended” feed screens resulted in consistently better product quality than the corresponding tighter or medium screen cassettes. The mass transfer coefficient for the lightly suspended screened channel was 260% higher than an open non-screened channel and only 22% lower than commercially available medium screen cassettes. The lightly suspended screen channel cassette thus offered the hydraulic advantage of lower differential pressure that an open channel offered, and the process advantage (processing time) that a medium screen channel offered along with consistently better product quality for the different biologics evaluated. The results of this study are useful in UF/DF process development of biologics to produce drug substance (DS) that has a better product quality and potentially lesser particulates.

The effect of self-aggregation on the determination of the kinetic and thermodynamic constants of the network of chemical reactions in 3-glucoside anthocyanins

The six most common 3-glucoside anthocyanins, pelargonidin-3-glucoside, peonidin-3-glucoside, delphinidin-3-glucoside, malvidin-3-glucoside, cyanidin-3-glucoside and petunidin-3-glucoside were studied in great detail by NMR, UV-vis absorption and stopped flow. For each anthocyanin, the thermodynamic and kinetic constants of the network of chemical reactions were calculated at different anthocyanin concentration, from 6 × 10⁻⁶ M up to 8 × 10⁻⁴ M; an increasing of the flavylium cation acidity constant to give quinoidal base and a decreasing of the flavylium cation hydration constant to give hemiketal were observed by increasing the anthocyanin concentration. These effects are attributed to the self-aggregation of the flavylium cation and quinoidal base, which is stronger in the last case. The UV-vis and ¹H NMR spectral variations resulting from the increasing of the anthocyanin concentration were discussed in terms of two aggregation models; monomer-dimer and isodesmic, the last one considering the formation of higher order aggregates possessing the same aggregation constant of the dimer. The self-aggregation constant of flavylium cation at pH=1.0, calculated by both models increases by increasing the number of methoxy (-OCH₃) or hydroxy (-OH) substituents following the order: myrtillin (2 -OH), oenin (2 -OCH₃), 3-OGl-petunidin (1 -OH, 1 -OCH₃), kuromanin (1 -OH), 3-OGl-peonidin (1 -OCH₃) and callistephin (none). Evidence for flavylium aggregates possessing a shape between J and H was achieved, as well as for the formation of higher order aggregates.

Concentration-Dependent Fluorescence Properties of Rhodamine 6G in Titanium Dioxide and Silicon Dioxide Nanolayers

Thin films of rhodamine 6G in titanium dioxide (Rh6G/TiO2) and silicon dioxide (Rh6G/SiO2) were synthesized using the sol–gel method. We explored two kinds of matrices as hosts for rhodamine 6G (Rh6G) at different concentrations of the dye. The pronounced effect of the dye concentration on the absorption and fluorescence spectra as well as on time-resolved fluorescence spectra was found. In particular, it was found that the aggregation of the guest dye is significantly weaker in the Rh6G/TiO2 nanolayer. The absence of an isosbestic point in absorption spectra in a silica matrix suggests the formation of higher order aggregates, whereas the changes of absorption profile in Rh6G/TiO2 matrix indicate the formation of dimers. Rh6G aggregates are strongly fluorescent in Rh6G/TiO2 nanolayer, which can be seen from time-resolved and steady-state emission spectra. For Rh6G/SiO2 nanolayers these changes are much less pronounced and concern mostly the red shift of the fluorescence maximum.

Enteric Commensal Bacteria Induce Extracellular Signal-regulated Kinase Pathway Signaling via Formyl Peptide Receptor-dependent Redox Modulation of Dual Specific Phosphatase 3

The normal microbial occupants of the mammalian intestine are crucial for maintaining gut homeostasis, yet the mechanisms by which intestinal cells perceive and respond to the microbiota are largely unknown. Intestinal epithelial contact with commensal bacteria and/or their products has been shown to activate noninflammatory signaling pathways, such as extracellular signal-related kinase (ERK), thus influencing homeostatic processes. We previously demonstrated that commensal bacteria stimulate ERK pathway activity via interaction with formyl peptide receptors (FPRs). In the current study, we expand on these findings and show that commensal bacteria initiate ERK signaling through rapid FPR-dependent reactive oxygen species (ROS) generation and subsequent modulation of MAP kinase phosphatase redox status. ROS generation induced by the commensal bacteria Lactobacillus rhamnosus GG and the FPR peptide ligand, N-formyl-Met-Leu-Phe, was abolished in the presence of selective inhibitors for G protein-coupled signaling and FPR ligand interaction. In addition, pretreatment of cells with inhibitors of ROS generation attenuated commensal bacteria-induced ERK signaling, indicating that ROS generation is required for ERK pathway activation. Bacterial colonization also led to oxidative inactivation of the redox-sensitive and ERK-specific phosphatase, DUSP3/VHR, and consequent stimulation of ERK pathway signaling. Together, these data demonstrate that commensal bacteria and their products activate ROS signaling in an FPR-dependent manner and define a mechanism by which cellular ROS influences the ERK pathway through a redox-sensitive regulatory circuit.

Process Monitoring the Inactivation of Ricin and Model Proteins by Disinfectants Using Fluorescence and Biological Activity

It is important to develop rapid and reliable processes to monitor the decontamination of toxins released to the environment. The inactivation of the protein toxin ricin by the disinfectants bleach (sodium hypochlorite) and monochloramine was measured by the effect on mammalian cell cytotoxicity. The effect of the disinfectants on the native fluorescence (due mainly to tryptophan and to a lesser extent tyrosine) of ricin was also measured in parallel. Reactions of the disinfectants resulted in a decrease in the native fluorescence that was measured in real time in a noninvasive manner. We compared the inactivation of two well-characterized model enzymes to the behavior of ricin. The model enzymes studied were lysozyme, a small basic enzyme stabilized with internal disulfide bonds, and heart-muscle-type lactate dehydrogenase (LDH), a large protein composed of four subunits. The biological activities of the model enzymes were measured in parallel with their fluorescence. Gel electrophoresis showed a large number of modifications of the proteins caused by the disinfectants reflected in changes in mobility and the formation of higher-order aggregates. Size-exclusion chromatography showed that the disinfectants did not break down the subunit structure of ricin but instead resulted in an increased size and heterogeneity of the protein. Size-exclusion chromatography of LDH indicated that the subunits were dissociated and that higher-order aggregates were also formed. Bleach caused a rapid inactivation of biological activity correlated with a rapid decrease in the fluorescence. Monochloramine required much higher concentrations for significant effects and the kinetics of the reactions were slow, with half-life values of the decrease on the order of minutes. Each protein showed individual differences in responses to the disinfectants, but there was a consistent correlation between the loss of fluorescence and the decrease in biological activity. These results indicate that the monitoring the fluorescence is a useful process with limitations that can be used to monitor the inactivation of toxins using disinfectants.

Aggregation behaviour of a dirhamnolipid biosurfactant secreted by Pseudomonas aeruginosa in aqueous media

The process of micelle formation, along with the formation of higher order aggregates, is described for a dirhamnolipid extracellular biosurfactant secreted by Pseudomonas aeruginosa. As determined by surface tension measurements, at pH 7.4 the CMC of dirhamnolipid is 0.110 mM, whereas at pH 4.0 it falls to 0.010 mM, indicating that the negatively charged diRL has a much higher CMC value than the neutral species. Centrifugation and dynamic light scattering measurements show formation of larger aggregates at concentrations above the CMC. These aggregates have been shown by electron microscopy to be mainly multilamellar vesicles of heterogeneous size. X-ray scattering gave a value of 32 Å for the interlamellar repeat distance of these vesicles. Taking into account the experimental data, a molecular modelling of the dirhamnolipid moiety has been carried out, which details the size of the hydrophilic and hydrophobic portions, and suggests the possible intermolecular interactions responsible for the stabilisation of dirhamnolipid aggregates. The relevance of this aggregation behaviour is discussed with respect to the molecular basis of its activities.

High Mobility of Carboxyl-Terminal Region of Bacterial Chemotaxis Phosphatase CheZ Is Diminished upon Binding Divalent Cation or CheY-P Substrate

In Escherichia coli chemotaxis, the CheZ phosphatase catalyzes the removal of the phosphoryl group from the signaling molecule, CheY. The cocrystal structure of CheZ with CheY x BeF3- x Mg2+ (a stable analogue of CheY-P) revealed that CheZ is a homodimer with a multidomain, nonglobular structure. To explore the effects of CheZ/CheY complex formation on CheZ structure, the rotational dynamics of the different structural domains of CheZ [the four-helix bundle, the N-terminal helix, the C-terminal helix, and the putative disordered linker between the C-terminal helix and the bundle] were evaluated. To monitor dynamics of the different regions, fluorescein probes were covalently attached at various locations on CheZ through reaction with engineered cysteine residues and the rotational behavior of the fluoresceinated derivatives were assessed using steady state fluorescence anisotropy. Anisotropy measurements at various solution viscosities (Perrin plot analysis) demonstrated large differences in global rotational motion for fluorophores located on different regions. Rotational correlation times for probes located on the four-helix bundle and the N-terminal helix agreed well with theoretical values predicted for a protein the size and shape of the four-helix bundle. However, the rotational correlation times of probes located on the linker and the C-terminal helix were 8-20x lower, indicating rapid motion independent of the bundle. The anisotropies of probes located on the linker and the C-terminal helix increased in the presence of divalent cation (Mg2+, Ca2+, or Mn2+) in a saturable fashion, consistent with a binding event (Kd approximately 1-4 mM) that results in decreased mobility. The anisotropies of probes located on the C-terminal helix and the C-terminal portion of the linker increased further as a result of binding CheY-P. In light of the recently available structural data and the high independent mobility of the C-terminus demonstrated here, we interpret the CheY-P-dependent increase in anisotropy to be a consequence of decreased mobility of the C-terminal region due to binding interactions with CheY-P, and not to the formation of higher order aggregates of the CheZ2(CheY-P)2 complex.

Spectral Properties, Formation of Dye Molecular Aggregates, and Reactions in Rhodamine 6G/Layered Silicate Dispersions

A series of layered silicates with a systematically modified charge was prepared from montmorillonite, using the standard method of Li+ cation fixation. The effect of the charge of the prepared materials on the optical properties of the adsorbed cationic dye, Rhodamine 6G, was tested. The layer charge of the silicate substrate controlled the formation of the dye's molecular assemblies, such as H-dimers and H- and J-aggregates. The dye species (monomers, aggregates) were characterized by distinctly different optical properties. Ultraviolet/visible and fluorescence spectroscopies were used for the characterization of the studied systems. Time difference spectroscopy was applied for the investigation of the reactions, which proceeded in dispersions after mixing the dye solution with silicate. The reactions included the formation of higher order aggregates at the surfaces of the high charge densities or dye deaggregation reactions at the surfaces of medium or low charge densities. Some trends and features of the R6G aggregation and the changes with time in relation to the layer charge are different from those that have been observed for other dyes in older studies. This may be due to the complicated structure and the specific shape of the R6G cation.

Organized assembly from biosurfactants

The formation of higher order aggregates in aqueous media from naturally occurring surfactants (biosurfactants) and its mechanism have been studied. A liposome (vesicle), as a kind of artificial cell, is prepared according to a newly developed microencapsulation technique from phospholipids by mimicking the molecular structure of biomembranes. Secondly, the surface active properties of alkaline salts of spiculisporic acid depend on the kinds of alkali as its gegen ions; these have been studied for the purpose of a detailed understanding of vesicle formation of the alkylamine salts of spiculisporic acid in the binary system consisting of spiculisporates and water. Thirdly, the solution behavior of the ternary system of spiculisporates-oil-water is examined. Divalent cationic salts of spiculisporic acid formed oil-in-water (O/W) emulsion gels due to ionic-bonding network formation of spiculisporates around emulsified oil droplets and these are important for their gelling properties. These gelling systems may have potential to be O/W emulsion-type paints and safe fuels.

Effect of self-association of α s1-casein and its cleavage fractions α s1-casein(136–196) and α s1-casein(1–197), on aromatic circular dichroic spectra: comparison with predicted models

The self-association of native α s1-casein is driven by a sum of interactions which are both electrostatic and hydrophobic in nature. The dichroism of aromatic side chains was used to derive regio-specific evidence in relation to potential sites of α s1-casein polymerization. Near-ultraviolet circular dichroism (CD) revealed that both tyrosine and tryptophan side chains play a role in α s1-casein associations. Spectral evidence shows these side chains to be in an increasingly nonaqueous environment as both ionic strength and protein concentration lead to increases in the degree of self-association of the protein from dimer to higher oligomers. Near-UV CD investigation of the carboxypeptidase A treated peptide, α s1-casein(1–197), indicated that the C-terminal residue (Trp199) may be superficial to these interactions, and that the region surrounding Trp164 is more directly involved in an aggregation site. Similar results for the cyanogen bromide cleavage peptide α s1-casein(136–196) indicated the presence of strongly hydrophobic interactions. Association constants for the peptides of interest were determined by analytical ultracentrifugation, and also were approximated from changes in the near-UV CD curves with protein concentration. Sedimentation equilibrium experiments suggest the peptide to be dimeric at low ionic strength; like the parent protein, the peptide further polymerizes at elevated (0.224 M) ionic strength. The initial site of dimerization is suggested to be the tyrosine-rich area near Pro147, while the hydrophobic region around Pro168, containing Trp164, may be more significant in the formation of higher-order aggregates.

Combined study of coagulation kinetics and close-range aggregate structure

The kinetics of coagulation of model polystyrene latex spheres under both reaction and diffusion control have been studied. The zeroth moment of the aggregate distribution has been measured by an automated laser particle-counting method, and the second moment by time-resolved low-angle light scattering. Emphasis has been both on dimer formation and the longer-time kinetics of cluster–cluster aggregation. Extension of the light scattering method of Giles and Lips (J. Chem. Soc., Faraday Trans. 1, 1974, 74, 733) has yielded information on the close-range structure in growing aggregates. The moments of the aggregate distribution can deviate from the predictions of the simple Smoluchowski constant-kernel equation. Acceleration in rate from reaction to diffusion control can occur in so-called rapid coagulation and in an intermediate regime of slow coagulation. In very slow coagulation, the rate decelerates, suggesting transition from reaction to equilibrium control. Interpretation on kinetic random polycondensation theory for an RAf process has yielded effective functionalities f which increase with particle density and decrease with increasing reaction control. The potential of spherical particles for high functional reactivity is not in general realised because aggregates can rearrange within the timescale of collisions, and spheres can form cycles (ring-closed structures) as early as the trimer stage, with consequent wastage of functionalities. The close-range structure of aggregates increases in compactness with increasing reaction control and decreasing particle concentration. The kinetics in the regime of the very slow coagulation are consistent with an initial reversible step of dimerisation followed by the irreversible formation of higher-order aggregates with ring-closed bonding. Application of reversible polycondensation theory to the dimer step has enabled the strength of attraction, a finite primary minimum, to be quantified. The observed deviations from constant-kernel Smoluchowski kinetics have important implications for the measurement of classical colloid stability plots and may be the origin of the discrepancies between the predictions of DLVO theory and kinetic measurements of colloid stability.

Aggregation of colloidal particles modeled as a dynamical process.

Aggregation kinetics of sonicated phosphatidylserine (PtdSer) vesicles in NaCl indicate that the process is fully reversible and dynamical, involving the rapid formation and dispersal of aggregates. Accordingly, the general mass action kinetic equations are analyzed with respect to the equilibrium state and the formation of higher order aggregates. For a general class of systems, the values for the mass average aggregate size at equilibrium are obtained from simple closed-form expressions. It is shown that an analysis of the aggregation equilibrium will yield estimates for the potential energy well that holds the aggregates together. A fit to the experimental data for kinetics of Na+-induced aggregation of the vesicles has been achieved by employing mass action kinetic equations that include the dissociation reactions. The threshold of NaCl concentration required for aggregation involves the clear distinction between the rate and extent of aggregation.