Transient Clusters Research Articles

Effect of Hierarchical Cluster Formation on the Viscosity of Concentrated Monoclonal Antibody Formulations Studied by Neutron Scattering.

Recently, reversible cluster formation was identified as an underlying cause of anomalously large solution viscosities observed in some concentrated monoclonal antibody (mAb) formulations, which poses a major challenge to the use of subcutaneous injection for some mAbs. A fundamental understanding of the structural and dynamic origins of high viscosities in concentrated mAb solutions is thus of significant relevance to mAb applications in human health care, as well as being of scientific interest. Herein, we present a detailed investigation of an IgG1-based mAb to relate the short-time dynamics and microstructure to significant viscosity changes over a range of pharmaceutically relevant physiochemical conditions. The combination of light scattering, small-angle neutron scattering, and neutron spin echo measurement techniques conclusively demonstrates that, upon addition of Na2SO4, these antibodies form strongly bound reversible dimers at dilute concentrations that interact with each other to form large, loosely bound, transient clusters when concentrated. This hierarchical structure formation in solution causes a significant increase in the solution viscosity.

Inorganic clusters with a [Fe2MoOS3] core-a functional model for acetylene reduction by nitrogenases.

We report the first example of a wholly inorganic mimic of a part of the FeMoco active centre of nitrogenases. We detail the synthesis, characterisation and reactivity of two related, transient hydride-containing inorganic clusters, a dihydride complex and a vinyl monohydride complex, which bear the [Fe2MoOS3] portion of FeMoco. The dihydride complex is capable of reducing acetylene to ethylene via the vinyl monohydride complex. In the reaction cycle, a transient low-valent complex was generated by the reductive elimination of H2 or ethylene from dihydride or vinyl monohydride complexes, respectively.

Discovery of Small Molecules that Inhibit the Disordered Protein, p27(Kip1).

Disordered proteins are highly prevalent in biological systems, they control myriad signaling and regulatory processes, and their levels and/or cellular localization are often altered in human disease. In contrast to folded proteins, disordered proteins, due to conformational heterogeneity and dynamics, are not considered viable drug targets. We challenged this paradigm by identifying through NMR-based screening small molecules that bound specifically, albeit weakly, to the disordered cell cycle regulator, p27Kip1 (p27). Two groups of molecules bound to sites created by transient clusters of aromatic residues within p27. Conserved chemical features within these two groups of small molecules exhibited complementarity to their binding sites within p27, establishing structure-activity relationships for small molecule:disordered protein interactions. Finally, one compound counteracted the Cdk2/cyclin A inhibitory function of p27 in vitro, providing proof-of-principle that small molecules can inhibit the function of a disordered protein (p27) through sequestration in a conformation incapable of folding and binding to a natural regulatory target (Cdk2/cyclin A).

Do Cyclodextrins Aggregate in Water? Insights from NMR Experiments.

One decade ago Bonini et al. [Langmuir 2006, 22, 1478-1484] reported the occurrence of aggregates of β-cyclodextrin in aqueous solutions with sizes in the range from 90 nm to a few micrometers. The experimental technique used was cryo-TEM. This work followed a number of previous studies involving other physical parameters, such as viscosities and activity coefficients, the results of which were interpreted in terms of self-aggregation of cyclodextrins. Since then, the ability of cyclodextrins to self-assemble were often used to explain and rationalize the supramolecular mechanisms involving cyclodextrins. Here, the question of aggregation of native cyclodextrins (α-, β-, and γ-) in aqueous solutions is addressed by using (1)H NMR techniques, including NMR diffusometry, relaxometry, and proton peak intensities. Within the detection limit of the NMR experiments, no aggregates of cyclodextrin were observed. If aggregates are present, the fraction of cyclodextrin in aggregates is quite small-less than 1%. However, we cannot exclude the presence of transient clusters involving several cyclodextrin molecules where the lifetime of the cluster is short.

Role of string-like collective atomic motion on diffusion and structural relaxation in glass forming Cu-Zr alloys.

We investigate Cu-Zr liquid alloys using molecular dynamics simulation and well-accepted embedded atom method potentials over a wide range of chemical composition and temperature as model metallic glass-forming (GF) liquids. As with other types of GF materials, the dynamics of these complex liquids are characterized by "dynamic heterogeneity" in the form of transient polymeric clusters of highly mobile atoms that are composed in turn of atomic clusters exhibiting string-like cooperative motion. In accordance with the string model of relaxation, an extension of the Adam-Gibbs (AG) model, changes in the activation free energy ΔGa with temperature of both the Cu and Zr diffusion coefficients D, and the alpha structural relaxation time τα can be described to a good approximation by changes in the average string length, L. In particular, we confirm that the strings are a concrete realization of the abstract "cooperatively rearranging regions" of AG. We also find coexisting clusters of relatively "immobile" atoms that exhibit predominantly icosahedral local packing rather than the low symmetry packing of "mobile" atoms. These two distinct types of dynamic heterogeneity are then associated with different fluid structural states. Glass-forming liquids are thus analogous to polycrystalline materials where the icosahedrally packed regions correspond to crystal grains, and the strings reside in the relatively disordered grain boundary-like regions exterior to these locally well-ordered regions. A dynamic equilibrium between localized ("immobile") and wandering ("mobile") particles exists in the liquid so that the dynamic heterogeneity can be considered to be type of self-assembly process. We also characterize changes in the local atomic free volume in the course of string-like atomic motion to better understand the initiation and propagation of these fluid excitations.

Two Classes of Cholesterol Binding Sites for the β2AR Revealed by Thermostability and NMR

Cholesterol binding to G protein-coupled receptors (GPCRs) and modulation of their activities in membranes is a fundamental issue for understanding their function. Despite the identification of cholesterol binding sites in high-resolution x-ray structures of the β2 adrenergic receptor (β2AR) and other GPCRs, the binding affinity of cholesterol for this receptor and exchange rates between the free and bound cholesterol remain unknown. In this study we report the existence of two classes of cholesterol binding sites in β2AR. By analyzing the β2AR unfolding temperature in lipidic cubic phase (LCP) as a function of cholesterol concentration we observed high-affinity cooperative binding of cholesterol with sub-nM affinity constant. In contrast, saturation transfer difference (STD) NMR experiments revealed the existence of a second class of cholesterol binding sites, in fast exchange on the STD NMR timescale. Titration of the STD signal as a function of cholesterol concentration provided a lower limit of 100 mM for their dissociation constant. However, these binding sites are specific for both cholesterol and β2AR, as shown with control experiments using ergosterol and a control membrane protein (KpOmpA). We postulate that this specificity is mediated by the high-affinity bound cholesterol molecules and propose the formation of transient cholesterol clusters around the high-affinity binding sites.

Absorbing phase transitions and dynamic freezing in running active matter systems.

We examine a two-dimensional system of sterically repulsive interacting disks where each particle runs in a random direction. This system is equivalent to a run-and-tumble dynamics system in the limit where the run time is infinite. At low densities, we find a strongly fluctuating state composed of transient clusters. Above a critical density that is well below the density at which non-active particles would crystallize, the system can organize into a drifting quiescent or frozen state where the fluctuations are lost and large crystallites form surrounded by a small density of individual particles. Although all the particles are still moving, their paths form closed orbits. The average transient time to organize into the quiescent state diverges as a power law upon approaching the critical density from above. We compare our results to the random organization observed for periodically sheared systems that can undergo an absorbing transition from a fluctuating state to a dynamical non-fluctuating state. In the random organization studies, the system organizes to a state in which the particles no longer interact; in contrast, we find that the randomly running active matter organizes to a strongly interacting dynamically jammed state. We show that the transition to the frozen state is robust against a certain range of stochastic fluctuations. We also examine the effects of adding a small number of pinned particles to the system and find that the transition to the frozen state shifts to significantly lower densities and arises via the nucleation of faceted crystals centered at the obstacles.

IgGs are made for walking on bacterial and viral surfaces.

Binding of antibodies to their cognate antigens is fundamental for adaptive immunity. Molecular engineering of antibodies for therapeutic and diagnostic purposes emerges to be one of the major technologies in combating many human diseases. Despite its importance, a detailed description of the nanomechanical process of antibody-antigen binding and dissociation on the molecular level is lacking. Here we utilize high-speed atomic force microscopy to examine the dynamics of antibody recognition and uncover a principle; antibodies do not remain stationary on surfaces of regularly spaced epitopes; they rather exhibit 'bipedal' stochastic walking. As monovalent Fab fragments do not move, steric strain is identified as the origin of short-lived bivalent binding. Walking antibodies gather in transient clusters that might serve as docking sites for the complement system and/or phagocytes. Our findings could inspire the rational design of antibodies and multivalent receptors to exploit/inhibit steric strain-induced dynamic effects.

Dynamical heterogeneity in periodically deformed polymer glasses

The dynamics of structural relaxation in a model polymer glass subject to spatially homogeneous, time-periodic shear deformation is investigated using molecular dynamics simulations. We study a coarse-grained bead-spring model of short polymer chains below the glass transition temperature. It is found that at small strain amplitudes, the segmental dynamics is nearly reversible over about 10^{4} cycles, while at strain amplitudes above a few percent, polymer chains become fully relaxed after a hundred cycles. At the critical strain amplitude, the transition from slow to fast relaxation dynamics is associated with the largest number of dynamically correlated monomers as indicated by the peak value of the dynamical susceptibility. The analysis of individual monomer trajectories showed that mobile monomers tend to assist their neighbors to become mobile and aggregate into relatively compact transient clusters.

In Silico Studies of Asymmetric Membranes Perturbations Caused by Dynamic Aggregation of a Cell-Penetrating Peptide

Membrane active peptides are therapeutically relevant for a variety of purposes. However a better understanding of their mechanisms of interaction with lipid bilayers is needed in order to maximise both efficiency and selectivity. In the case of cell-penetrating peptides (CPP) it is particularly important to avoid membrane toxicity while maintaining translocation across the plasma membrane. Experimental in vitro studies based on light microscopy and dye release have shown CPP can be internalised via both endocytic and energy-independent pathways. However, uncertainties remain concerning the mechanisms involved in membrane translocation and perturbation. In silico studies using molecular dynamics (MD) simulations have hitherto mainly focused on the interactions of peptides with relatively simple lipid bilayer models.Here we present coarse-grained simulations of the interactions between transportan, a CPP known to perturb cell membranes, and large bilayers with biologically relevant lipid composition. We observe that transportan forms dynamic, unstructured and transient clusters that catalyse the formation of local defects such as bilayer thinning, lipid redistribution and decrease of the lipid tail order. We present a novel analytic approach which shows that the extent of the membrane perturbations induced by CPP clusters depends on their size and varies in time. In particular, anionic lipid flip-flops are consistently observed above a certain cluster size. The importance of asymmetric lipid composition in the bilayer is also investigated and found to impact the stability of the peptide aggregates. We also apply our approach to extended bilayer systems that contain approximately 50,000 lipids and hundreds of transportan peptides and thus allow comparison with high-resolution light microscopy results.

Collective dynamics of flowing colloids during pore clogging.

Based on direct numerical simulations of the coupled motion of particles and fluid, this study analyzes the collective hydrodynamic and colloidal effects of flowing microparticles during the formation of different 3D clogging patterns at a pore entrance. Simulations of flowing suspensions through a pore with various simulation conditions show that particle concentration and surface interactions play a major role in the occurrence of the bridging phenomenon (simultaneous adhesion of many particles). In the absence of DLVO repulsive forces, plugging is characterized by the temporal reduction of the bulk permeability when increasing the volume fraction of the flowing suspension up to 20%. Under these conditions, different structures of particle aggregates (from cluster to cake plug) are formed at the pore entrance yielding different evolution rates of hydrodynamic resistance to the flow. Taking into account DLVO repulsive forces in simulations for a particle concentration equal to 10%, we observe the transition from dendritic structures (for low repulsion) to dense aggregates (for high repulsion). At high DLVO repulsive forces, the scenario of pore clogging is controlled by the collective behavior of many interacting particles. It leads to the formation of a jamming phase (Wigner glass phase) with transient clusters of interacting particles at the pore entrance. The network of jammed particles collapses when the force chains among the particles are overcome by the flow stress. The build-up and the collapse of the jammed phase at the pore entrance induce temporal permeability fluctuations. According to the relative magnitude of particle-particle and particle-wall interactions, when the jammed phase is disorganized by the flow, the residual force in the network can accelerate particles and lead to particle adhesion at the wall inducing a pore blockage and a rapid reduction of the bulk permeability.

GDI-Mediated Cell Polarization in Yeast Provides Precise Spatial and Temporal Control of Cdc42 Signaling

Cell polarization is a prerequisite for essential processes such as cell migration, proliferation or differentiation. The yeast Saccharomyces cerevisiae under control of the GTPase Cdc42 is able to polarize without the help of cytoskeletal structures and spatial cues through a pathway depending on its guanine nucleotide dissociation inhibitor (GDI) Rdi1. To develop a fundamental understanding of yeast polarization we establish a detailed mechanistic model of GDI-mediated polarization. We show that GDI-mediated polarization provides precise spatial and temporal control of Cdc42 signaling and give experimental evidence for our findings. Cell cycle induced changes of Cdc42 regulation enhance positive feedback loops of active Cdc42 production, and thereby allow simultaneous switch-like regulation of focused polarity and Cdc42 activation. This regulation drives the direct formation of a unique polarity cluster with characteristic narrowing dynamics, as opposed to the previously proposed competition between transient clusters. As the key components of the studied system are conserved among eukaryotes, we expect our findings also to apply to cell polarization in other organisms.

Different Kinetic Pathways of Early Stage Calcium-Phosphate Cluster Aggregation Induced by Carboxylate-Containing Polymers

Acidic proteins are critical to biomineral formation, although their precise mechanistic function remains poorly understood. A number of recent studies have suggested a nonclassical mineralization model that emphasizes the importance of the formation of polymer-stabilized mineral clusters or particles; however, it has been difficult to characterize the precursors experimentally due to their transient nature. Here, we successfully captured stepwise evolution of transient CaP clusters in mineralizing solutions and studied the roles of functional polymers with laser light scattering (LLS) to determine how these polymers influence the stability of nanoclusters. We found that the polymer structure can alter CaP aggregation mechanisms, whereas the polymer concentration strongly influences the rate of CaP aggregation. Our results indicate that the ability of acidic biomolecules to control the formation of relatively stable nanoclusters in the early stages may be critical for intrafibrillar mineralization. More importantly, LLS provided information about the size and the structural evolution of CaP aggregates, which will help define the process of controlled biomineralization.

The spatial structure of cell signaling systems

The spatial structure of the cell is highly organized at all levels: from small complexes and assemblies, to local nano- and microclusters, to global, micrometer scales across and between cells. We suggest that this multiscale spatial cell organization also organizes signaling and coordinates cellular behavior. We propose a new view of the spatial structure of cell signaling systems. This new view describes cell signaling in terms of dynamic allosteric interactions within and among distinct, spatially organized transient clusters. The clusters vary over time and space and are on length scales from nanometers to micrometers. When considered across these length scales, primary factors in the spatial organization are cell membrane domains and the actin cytoskeleton, both also highly dynamic. A key challenge is to understand the interplay across these multiple scales, link it to the physicochemical basis of the conformational behavior of single molecules and ultimately relate it to cellular function. Overall, our premise is that at these scales, cell signaling should be thought of not primarily as a sequence of diffusion-controlled molecular collisions, but instead transient, allostery-driven cluster re-forming interactions.

Chemotaxis of Dictyostelium discoideum: Collective Oscillation of Cellular Contacts

Chemotactic responses of Dictyostelium discoideum cells to periodic self-generated signals of extracellular cAMP comprise a large number of intricate morphological changes on different length scales. Here, we scrutinized chemotaxis of single Dictyostelium discoideum cells under conditions of starvation using a variety of optical, electrical and acoustic methods. Amebas were seeded on gold electrodes displaying impedance oscillations that were simultaneously analyzed by optical video microscopy to relate synchronous changes in cell density, morphology, and distance from the surface to the transient impedance signal. We found that starved amebas periodically reduce their overall distance from the surface producing a larger impedance and higher total fluorescence intensity in total internal reflection fluorescence microscopy. Therefore, we propose that the dominant sources of the observed impedance oscillations observed on electric cell-substrate impedance sensing electrodes are periodic changes of the overall cell-substrate distance of a cell. These synchronous changes of the cell-electrode distance were also observed in the oscillating signal of acoustic resonators covered with amebas. We also found that periodic cell-cell aggregation into transient clusters correlates with changes in the cell-substrate distance and might also contribute to the impedance signal. It turned out that cell-cell contacts as well as cell-substrate contacts form synchronously during chemotaxis of Dictyostelium discoideum cells.

Antibody Movement on Regular Antigen Clusters: Fab Arms are made for Walking

Antibodies are key molecules for the immune system of vertebrates. The Y-shaped IgGs exhibit C2-symmetry; their Fc stem is connected to two identical Fab arms binding antigens. The Fc part is recognized by the complement system and by phagocytic cells. Antibodies can be considered molecular calipers; bivalent binding of the two Fab arms to adjacent antigens can only occur within a distance of roughly 6 to 12 nm. This leads to much higher avidity and slower dissociation rates as compared to monovalent binding. Here we show that antibodies exhibit “bipedal” walking on antigenic surfaces and static binding of both Fab arms of an antibody may hold true only for a time scale of ∼ 0.04 s. The walking speed depends on the lateral spacing and symmetry of the antigens. On 2D-crystalline surfaces, such as found on bacteria and viruses, steric strain thus appears to be the main reason for short-lived bivalent binding. Importantly, the collision between randomly walking antibodies was seen to reduce their motional freedom. It leads to formation of transient antibody clusters even at low antibody density. Interestingly, such assemblies are known nucleation sites for docking of the complement system and/or phagocytes.

Hydration and Mobility of Trehalose in Aqueous Solution

The disaccharide trehalose stabilizes proteins against unfolding, but the underlying mechanism is not well understood. Because trehalose is preferentially excluded from the protein surface, it is of interest to examine how trehalose modifies the structure and dynamics of the solvent. From the spin relaxation rates of deuterated trehalose and (17)O-enriched water, we obtain the rotational dynamics of trehalose and water in solutions over wide ranges of concentration (0.025-1.5 M) and temperature (236-293 K). The results reveal direct solute-solute interactions at all concentrations, consistent with transient trehalose clusters. Similar to other organic solutes, the trehalose perturbation of water rotation (and hydrogen-bond exchange) is modest: a factor 1.6 (at 298 K) on average for the 47 water molecules in the first hydration layer. The deviation of the solute tumbling time from the Stokes-Einstein-Debye relation is partly caused by a dynamic solvent effect that is often modeled by incorporating "bound water" in the hydrodynamic volume. By comparing the measured temperature dependences of trehalose and water dynamics, we demonstrate that a more realistic local viscosity model accounts for this second-order dynamic coupling.

Active Remodeling of Cortical Actin Regulates Spatiotemporal Organization of Cell Surface Molecules

Many lipid-tethered proteins and glycolipids exist as monomers and nanoclusters on the surface of living cells. The spatial distribution and dynamics of formation and breakup of nanoclusters does not reflect thermal and chemical equilibrium and is controlled by active remodeling of the underlying cortical actin. We propose a model for nanoclustering based on active hydrodynamics, wherein cell surface molecules bound to dynamic actin are actively driven to form transient clusters. This consistently explains all of our experimental observations. Using FCS and TIRF microscopy, we provide evidence for the existence of short, dynamic, polymerizing actin filaments at the cortex, a key assumption of the theoretical framework. Our theory predicts that lipid-anchored proteins that interact with dynamic actin must exhibit anomalous concentration fluctuations, and a cell membrane protein capable of binding directly to actin can form nanoclusters. These we confirm experimentally, providing an active mechanism for molecular organization and its spatiotemporal regulation on the plasma membrane.

How Péclet number affects microstructure and transient cluster aggregation in sedimenting colloidal suspensions

We study how varying the Péclet number (Pe) affects the steady state sedimentation of colloidal particles that interact through short-ranged attractions. By employing a hybrid molecular dynamics simulation method we demonstrate that the average sedimentation velocity changes from a non-monotonic dependence on packing fraction φ at low Pe numbers, to a monotonic decrease with φ at higher Pe numbers. At low Pe number the pair correlation functions are close to their equilibrium values, but as the Pe number increases, important deviations from equilibrium forms are observed. Although the attractive forces we employ are not strong enough to form permanent clusters, they do induce transient clusters whose behaviour is also affected by Pe number. In particular, clusters are more likely to fragment and less likely to aggregate at larger Pe numbers, and the probability of finding larger clusters decreases with increasing Pe number. Interestingly, the lifetime of the clusters is more or less independent of Pe number in the range we study. Instead, the change in cluster distribution occurs because larger clusters are less likely to form with increasing Pe number. These results illustrate some of the subtleties that occur in the crossover from equilibrium like to purely non-equilibrium behaviour as the balance between convective and thermal forces changes.

Cluster formation in star-linear polymer mixtures: equilibrium and dynamical properties

We investigate the influence of the addition of polymer chains on suspensions of low-functionality star polymers in the dilute star regime. We focus on the structural and dynamical features of the mixture by means of Molecular Dynamic simulations, based on a recently introduced model, which coarse-grained the interactions between the two components of the system. Numerical results are compared to the analytical ones, obtained from mode-coupling theory. We find evidence for the formation of transient star clusters upon chain addition, which is induced by the depletion attraction between the stars. Contrary to the case of hard colloids with a depletion-induced short-range attraction and a long range repulsion, the formed clusters are irregular and transient, allowing for particle exchange among themselves.