Early Stages of FUS Droplet Formation via Liquid-Liquid Phase Separation.

The phase behavior of complex biomolecular solutions may explain different cellular processes, including the organization of cells by membraneless organelles. The early stages of phase separation are crucial to understanding the underlying mechanism and identifying biomolecules that trigger or drive the transition. Here, we analyze the early events of liquid-liquid phase separation (LLPS) of FUS by multiangle time-resolved static and dynamic light scattering. LLPS was triggered by TEV-catalyzed cleavage of the MBP-tag from FUS-MBP. The light scattering measurements revealed the existence of at least two fractions of FUS-MBP aggregates already prior to the onset of LLPS. The orders of magnitude of the aggregate size in these two fractions are 10 and 100 nm, respectively. LLPS started after an induction period, which depended on the concentration of FUS-MBP. The data from time-dependent light scattering revealed a coalescence of droplets also denoted as a step growth process. A step growth process instead of nucleation and growth via monomer addition suggests that LLPS takes place within the spinodal rather than between the binodal and the spinodal.

Impact of Additive Hydrophilicity on Mixed Dye-Nonionic Surfactant Micelles: Micelle Morphology and Dye Localization.

The nonionic surfactant pentaethylene glycol-monododecylether C12E5 forms micelles in aqueous solutions with a lower critical solution temperature. This characteristic solution behavior of C12E5 is independent of the pH. Such micelles are used to solubilize a large variety of active guest molecules like for instance dyestuffs. An example is an acidic azo dye termed Blue used as a hair colorant. Depending on the pH, Blue gradually changes its hydrophilicity from the protonated BlueH at pH = 2 to the bivalent anion Blue2- at pH = 13 while keeping the shape and size of Blue essentially unchanged. These features of C12E5 and Blue offer the unique chance to investigate the sole impact of a tunable hydrophilicity of a guest molecule on the solution behavior of mixed micelles of the guest and C12E5. Accordingly, the present work establishes a phase diagram of Blue-C12E5 micelles and analyzes their morphology including the spatial distribution of Blue in the micelles as a function of the hydrophilicity of Blue. Small angle neutron scattering reveals the size and shape of the micelles, and detailed contrast matching of the C12E5 supported by 1H NMR with NOESY provided insight into the localization of Blue within the micelles as its hydrophilicity changes.

Molecular Crowding: The History and Development of a Scientific Paradigm.

It is now generally accepted that macromolecules do not act in isolation but "live" in a crowded environment, that is, an environment populated by numerous different molecules. The field of molecular crowding has its origins in the far 80s but became accepted only by the end of the 90s. In the present issue, we discuss various aspects that are influenced by crowding and need to consider its effects. This Review is meant as an introduction to the theme and an analysis of the evolution of the crowding concept through time from colloidal and polymer physics to a more biological perspective. We introduce themes that will be more thoroughly treated in other Reviews of the present issue. In our intentions, each Review may stand by itself, but the complete collection has the aspiration to provide different but complementary perspectives to propose a more holistic view of molecular crowding.

On the Mechanism of Self-Assembly of Fibrinogen in Thrombin-free Aqueous Solution.

Fibrinogen dissolved in 0.12 M aqueous NaCl solution at a pH of 6.6 exhibits self-assembly in response to a lowering of the NaCl concentration to values equal to or lower than 60 mM. As has been established in a preceding work (Langmuir 2019, 35, and 12113), a characteristic signature of the self-assembly triggered by a drop in ionic strength is the formation of large globular particles. Growth of these particles most likely obeys a coalescence-like process also termed a step growth process. In order to extend this knowledge, the present work first optimized the protocol, leading to highly reproducible self-assembly experiments. Based on this optimization, the work succeeded in identifying an initial stage, not yet accessible, during which rigid short fibrils grow in close analogy to the thrombin-catalyzed polymerization of fibrin. In addition, first suggestions could be made on the transformation of these fibrils into larger aggregates, which upon drying turn into thick fiber-like ropes.

SANS contrast matching for the unambiguous localization of anionic dye in cationic surfactant micelles.

Contrast variation in small-angle neutron scattering (SANS) was successfully applied to localize the anionic azo dye Blue in co-assemblies with the cationic surfactant dodecyltrimethylammoniumbromide (DTAB). For this purpose, the scattering contrast between DTAB and the aqueous solvent was eliminated by SANS contrast matching, leaving only the scattering signal from Blue to be detected. Results obtained by contrast matching were confirmed by NOESY NMR-spectroscopy, showing that Blue interacts with the positively charged DTAB head groups and with up to the 4th neighbouring methylene group of the DTAB C12-alkyl chain. Its localization in the outer layer of the Blue-DTAB co-assembly explains the uniaxial growth of spheroidal DTAB micelles to wormlike micelles with increasing [Blue] : [DTAB] ratio from 0 : 1 to 1 : 3. This is in line with the concept of the packing parameter for amphiphilic substances.

Ethylene glycol energetically disfavours oligomerization of pseudoisocyanine dyestuffs at crowded concentrations.

The intriguing role of the intracellular crowded environment in regulating protein aggregation remains elusive. The convolution of several factors such as the protein sequence-dependence, crowder's shape and size and diverse intermolecular interactions makes it complex to identify systematic trends. One of the ways to simplify the problem is to study a synthetic model for self-assembling proteins. In this study, we examine the aggregation behaviour of the cationic pseudoisocyanine chloride (PIC) dyestuff which is known to self-assemble and form fibril-like J-aggregates in aqueous solutions, similar to those formed by amyloid-forming proteins. Prior experimental studies have shown that polyethylene glycol impedes and Ficoll-400 promotes the self-assembly of PIC dyes. To achieve molecular insights, we examine the effect of crowding by ethylene glycol on the solvation thermodynamics of oligomerization of dyes into H-type and J-type oligomers using extensive molecular dynamics simulations. The binding free energy calculations show that the formation of J-oligomers is more favourable than that of H-oligomers in water. The stability of H- and J- tetramers and pentamers decreases in crowded solutions. The formation of oligomers is supported by the favourable change in dye-solvent interaction energy in both pure water and aqueous ethylene glycol solution although it is opposed by the reduced dye-solvent entropy. Ethylene glycol, as a molecular crowder, disfavours the H- as well as J-oligomerization via preferential binding to the dye oligomers. An unfavourable change in dye-crowder and dye-dye interaction energy on dye association makes the H-oligomer formation less favourable in crowded solution than in pure water solution. In the case of J-oligomers, however, the unfavourable change in dye-crowder interaction energy primarily contributes to making total dye-solvent energy unfavourable. The results are supported by isothermal titration calorimetry measurements where the binding of ethylene glycol to PIC molecules is found to be endothermic. The results provide an emerging view that a crowded environment can disfavour self-assembly of PIC dyes by interactions with the oligomeric states. The findings have implications in understanding the role of a crowded environment in shaping the free energy landscapes of proteins.

A Comparative Study on Cyanine Dyestuffs as Sensor Candidates for Macromolecular Crowding In Vitro and In Vivo.

Pseudo isocyanine chloride (PIC) has been identified in a preceding work as a sensor suited to probe macromolecular crowding both in test tubes with solutions of synthetic crowding agents and in HeLa cells as a representative of living systems. The sensing is based on a delicate response of the self-assembly pattern of PIC towards a variation in macromolecular crowding. Based on a suitable selection of criteria established in the present study, four additional cyanine dyestuffs (TDBC, S071, S2275, and PCYN) were scrutinized for their ability to act as such a sensor, and the results were compared with the corresponding performance of PIC. UV-VIS and fluorescence spectroscopy were applied to investigate the photo-physical properties of the four candidates and, if possible, light scattering was used to characterize the self-assembly of the dyestuffs in solution. Finally, HeLa cells were exposed to solutions of the most promising candidates in order to analyze their ability to infiltrate the cells and to self-assemble therein. None of the dyestuff candidates turned out to be as similarly promising in probing crowding effects in cells as PIC turned out to be. S0271 and S2275 are at least stable enough and meet the photophysical requirements necessary to act as sensors responding to changes in macromolecular crowding.

Comparative study of the self-assembly behaviour of 3-chloro-4-hydroxy-phenylazo dyes.

The complexity of intermolecular interactions and the difficulty to predict assembly behaviour solely based on chemical constitution was demonstrated by studying the self-assembly of three one-fold negatively charged 3-chloro-4-hydroxy-phenylazo dyes (Yellow, Blue and Red). Dye self-assembly was investigated using UV/vis- and NMR-spectroscopy, light- and small-angle neutron scattering. Significant differences between the three dyes were observed. While Yellow does not self-assemble, Red assembles into higher-order aggregates and Blue forms well-defined H-aggregate dimers with a dimerization constant of KD = (728 ± 8) L mol-1. Differences between dyes were suggested to emerge from variations in the propensity to form π-π-interactions due to electrostatic repulsion, sterical constraints and hydrogen-bonding interactions.

Comparative study of the co-assembly behaviour of 3-chloro-4-hydroxy-phenylazo dyes with DTAB.

The co-assembly of three one-fold negatively charged 3-chloro-4-hydroxy-phenylazo dyes (Yellow, Blue and Red) with the cationic surfactant dodecyltrimethylammoniumbromide (DTAB) was studied to probe dye-DTAB binding stoichiometry and assembly morphology. For each dye, phase separation was observed above a given dye : DTAB ratio with the ratio depending on the dye. While Yellow and DTAB showed liquid/liquid phase separation above Yellow : DTAB = 1 : 1.67, crystalline dye-DTAB complexes were observed for Blue-DTAB and Red-DTAB above Blue : DTAB = 1 : 2.56 and Red : DTAB = 1 : 2.94 respecively. In homogeneous solution, UV/vis spectroscopic investigations suggest stochiometries of Yellow : DTAB = 1 : 2, Blue : DTAB = 1 : 3 and Red : DTAB = 1 : 4. It was concluded, that Yellow exhibits the highest dye : DTAB binding stoichiometry in both, dye-surfactant complexes in the 2-phase region and in solution, whereas the lowest dye : DTAB binding stoichiometry was observed for Red-DTAB in both cases. The observed stoichiometries are inversely correlated to the impact dye addition has on the morphology of DTAB micelles. Generally, addition of dye to DTAB micelles leads to a reduction in spontaneous curvature of these micelles and to the formation of triaxial ellipsoidal or cylindrical micelles from oblate ellipsoidal DTAB micelles. At a DTAB concentration of 30 mM and a dye concentration of 5 mM, this effect was most pronounced for Red and least pronounced for Yellow, whilst Blue showed an intermediate effect.

Spatial Distribution of Intracellular Ion Concentrations in Aggregate-Forming HeLa Cells Analyzed by µ-XRF Imaging.

Protein aggregation is a hallmark of several severe neurodegenerative disorders such as Huntington's, Parkinson's, or Alzheimer's disease. Metal ions play a profound role in protein aggregation and altered metal-ion homeostasis is associated with disease progression. Here we utilize µ-X-ray fluorescence imaging in combination with rapid freezing to resolve the elemental distribution of phosphorus, sulfur, potassium, and zinc in huntingtin exon-1-mYFP expressing HeLa cells. Using quantitative XRF analysis, we find a threefold increase in zinc and a 10-fold enrichment of potassium that can be attributed to cellular stress response. While the averaged intracellular ion areal masses are significantly different in aggregate-containing cells, a local intracellular analysis shows no different ion content at the location of intracellular inclusion bodies. The results are compared to corresponding experiments on HeLa cells forming pseudoisocyanine chloride aggregates. As those show similar results, changes in ion concentrations are not exclusively linked to huntingtin exon-1 amyloid formation.

Targeted Synthesis of the Type-A Particle Substructure from Enzymatically Produced Eumelanin.

Eumelanin exhibits a defined supramolecular buildup that is deprived of at least three distinct particle species. To enable the full potential of its promising material properties, access to all particle types is crucial. In this work, the first protocol for the synthesis of the intermediate type-A particles in pure and stable dispersion form is described. It is found that aggregation of type-A particles into the larger type-B variant can be inhibited by a strict pH control during the synthesis. The exact influence of pH on the supramolecular buildup is investigated via a combination of time-resolved light scattering, electron microscopy, and UV-vis spectroscopy. It is observed that a rapid buildup of type-B particles occurs without pH control and is generally dominant at lower pH values. At pH values above 6.2 however, type-A particles are gained, and no further aggregation occurs. Even more, lowering the pH of such a stable type-A dispersion at a later stage lifts the inhibition and again leads to the formation of larger particle species. The results confirm that it is easily possible to halt the aggregation of eumelanin substructures and to access them in the form of a stable dispersion. Moreover, a profound additional understanding of the supramolecular buildup is gained by the in-depth investigation of the pH influence.

Mechanism and equilibrium thermodynamics of H- and J-aggregate formation from pseudo isocyanine chloride in water.

Pseudo isocyanine chloride (PIC) has a strong tendency to self-assemble with a concentration dependent temperature threshold separating a regime with small H-oligomers in equilibrium with monomeric PIC from a regime where large J-aggregates form. In complementing the known set of absorption spectra by the spectrum of a trimer, which represents all H-aggregates with N ≥ 3, a full description of the sample composition of PIC in the regime of oligomers became possible by means of UV-vis spectroscopy and gave access to the equilibrium thermodynamics of oligomerisation. Successful interpretation of the concentration dependent temperature threshold as a ceiling temperature of J-aggregation made also accessible equilibrium thermodynamics of the formation of J-aggregates together with a full analysis of composition also in the regime of J-aggregates. Evolution of an invariant spectrum for the J-aggregates demonstrates full consistency of the composition analysis. Complementary light scattering experiments led to a comprehensive characterisation of all aggregate species above and below the aggregation threshold. Once initiated, J-aggregates always grow to a final length of 650 nm. Time-resolved light scattering confirmed that the self-assembly of J-aggregates follows a monomer addition process in analogy to a chain growth in polymer chemistry. Initiation and growth of individual aggregates turned out to be always much faster than the progress of aggregation.

Self-Assembled Fibrinogen Hydro- and Aerogels with Fibrin-like 3D Structures.

The natural blood protein fibrinogen is a highly potent precursor for the production of various biomaterials due to its supreme biocompatibility and cell interaction. To gain actual materials from fibrinogen, the protein needs to undergo fibrillogenesis, which is mostly triggered via enzymatic processing to fibrin, electrospinning, or drying processes. All of those techniques, however, strongly limit the available structures or the applicability of the material. To overcome the current issues of fibrin(ogen) as material, we herein present a highly feasible, quick, and inexpensive technique for self-assembly of fibrinogen in solution into defined, nanofibrous three-dimensional (3D) patterns. Upon interaction with specific anions in controlled environments, stable and flexible hydrogel-like structures are formed without any further processing. Moreover, the material can be converted into highly porous and elastic aerogels by lyophilization. Both of these material classes have never been described before from native fibrinogen. The observed phenomenon also represents the first enzyme-free process of fibrillogenesis from fibrinogen with significant yield in solution. The produced hydrogels and aerogels were investigated via electron microscopy, IR spectroscopy, and fluorescence spectroscopy, which also confirms the native state of the protein. Additionally, their mechanical properties were compared with actual fibrin and unstructured fibrinogen. The structural features show a striking analogy to actual fibrin, both as hydro- and aerogel. This renders the new material a highly promising alternative for fibrin in biomaterial applications. A much faster initiation of fiber formation, exclusion of possible thrombin residuals, and low-cost reagents are great advantages.

Self-Assembly of Pseudo-Isocyanine Chloride as a Sensor for Macromolecular Crowding In Vitro and In Vivo.

Pseudo-isocyanine chloride (PIC) is a cationic dyestuff that exhibits self-assembly in aqueous solution, promoted either by increasing the PIC concentration or by decreasing the temperature. PIC-aggregates exhibit a characteristic and sharp absorption band as well as a fluorescence band at a wavelength of 573 nm making PIC an interesting candidate to analyze the self-assembly process in various environments. The present work developed PIC-based, synthetic model systems, suitable to investigate how macromolecular crowding influences self-assembly processes. Four synthetic additives were used as potential crowders: Triethylene glycol (TEG), polyethylene glycol (PEG), Ficoll 400 as a highly branched polysaccharide, and sucrose corresponding to the monomeric unit of Ficoll. Combined UV/Vis spectroscopy and time-resolved light scattering revealed a strong impact of crowding based on excluded volume effects only for Ficoll 400. Sucrose had hardly any influence on the self-assembly of PIC and PEG and TEG impeded the PIC self-assembly. Development of such a PIC based model system led over to in-cell experiments. HeLa cells were infiltrated with PIC solutions well below the aggregation threshold in the infiltrating solution. In the cellular environment, PIC was exposed to a significant crowding and immediately started to aggregate. As was demonstrated by fluorescence imaging, the extent of aggregation can be modulated by exposing the cells to salt-induced osmotic stress. The results suggest future use of such a system as a sensor for the analysis of in vitro and in vivo crowding effects on self-assembly processes.

The Molecular Mechanism of Polymer Formation of Farnesylated Human Guanylate-binding Protein 1.

The human guanylate-binding protein 1 (hGBP1) belongs to the dynamin superfamily proteins and represents a key player in the innate immune response. Farnesylation at the C-terminus is required for hGBP1's activity against microbial pathogens, as well as for its antiproliferative and antitumor activity. The farnesylated hGBP1 (hGBP1fn) retains many characteristics of the extensively studied nonfarnesylated protein and gains additional abilities like binding to lipid membranes and formation of hGBP1fn polymers. These polymers are believed to serve as a protein depot, making the enzyme immediately available to fight the invasion of intracellular pathogens. Here we study the molecular mechanism of hGBP1 polymer formation as it is a crucial state of this enzyme, allowing for a rapid response demanded by the biological function. We employ Förster resonance energy transfer in order to trace intra and intermolecular distance changes of protein domains. Light scattering techniques yield deep insights into the changes in size and shape. The GTP hydrolysis driven cycling between a closed, farnesyl moiety hidden state and an opened, farnesyl moiety exposed state represents the first phase, preparing the molecule for polymerization. Within the second phase of polymer growth, opened hGBP1 molecules can be incorporated in the growing polymer where the opened structure is stabilized, similar to a surfactant molecule in a micelle, pointing the farnesyl moieties into the hydrophobic center and positioning the head groups at the periphery of the polymer. We contribute the molecular mechanism of polymer formation, paving the ground for a detailed understanding of hGBP1 function.

Controlling Self-Assembly with Light and Temperature.

Complexes between the anionic polyelectrolyte sodium polyacrylate (PA) and an oppositely charged divalent azobenzene dye are prepared in aqueous solution. Depending on the ratio between dye and polyelectrolyte stable aggregates with a well-defined spherical shape are observed. Upon exposure of these complexes to UV light, the trans → cis transition of the azobenzene is excited resulting in a better solubility of the dye and a dissolution of the complexes. The PA chains reassemble into well-defined aggregates when the dye is allowed to relax back into the trans isomer. Varying the temperature during this reformation step has a direct influence on the final size of the aggregates rendering temperature in an efficient way to easily change the size of the self-assemblies. Application of time-resolved small-angle neutron scattering (SANS) to study the structure formation reveals that the cis → trans isomerization is the rate-limiting step followed by a nucleation and growth process.

On Protein Folding in Crowded Conditions.

The interior of a cell is a highly packed environment that can be occupied up to 40% by different macromolecules. Such crowded media influence different biochemical processes like protein folding, enzymatic activity, and gene regulation. In this work, we use simulations to study protein stability under the presence of crowding agents that interact with the protein by excluded volume interactions. In general, the presence of crowding agents in the solution enhances the stability of the protein's native state. However, we find that the effects of excluded volume depend not only on crowding occupancy but also the crowders' geometry and size. Specifically, we find that polymeric crowders have stronger influence than spherical crowders and that this effect increases with polymer length, while it decreases with increasing size of spherical crowders. These opposing size effects are explained by the interplay of decreasing excluded volume and demixing, which together determine the change in the entropy of the crowders upon folding of the protein.

Ion-selective binding as a new trigger for micellization of block copolyelectrolytes with two anionic blocks.

This work presents well-defined and switchable micelles of block copolymers consisting of the two anionic polyelectrolytes sodium polyacrylate (NaPA) and sodium polystyrene sulfonate (NaPSS). Micellization occurs due to the specific binding of Ca2+ to acrylate groups, which results in neutralization of the corresponding block and thereby formation of the hydrophobic core of the micelles. In contrast, the PSS block remains charged and forms the stabilizing shell. Micellization is triggered by variations of the Ca2+ concentration or the temperature and is a fully reversible and repeatable process. Small-angle neutron scattering (SANS) could unambiguously reveal the structure of the micelles, using a partially deuterated polymer and the contrast variation technique. Considering the variety of metal cations and their broad spectrum of interactions with polyelectrolytes, this new class of like-charged block copolymers opens the door to a broad range of switchable and responsive polyelectrolyte-based systems.

Self-Assembly of Fibrinogen in Aqueous, Thrombin-Free Solutions of Variable Ionic Strengths.

Fibrinogen not only forms fibrin networks if assisted by thrombin but also exhibits self-assembly in dilute aqueous solutions in the absence of thrombin. It could be shown that self-assembly can be triggered in a controlled way by diluting the ionic strength set to a value of 0.14 M NaCl in the starting solutions. The present work unravels the mechanism of this self-assembly process by means of a combination of time-resolved multiangle static and dynamic light scattering and atomic force microscopy. Analysis was carried out as a function of the ionic strength adjusted by the drop in ionic strength and at variable salt compositions at a given final ionic strength. Composition was varied by changing the ratio of NaCl and phosphate buffer. The self-assembly induced by the drop of the ionic strength depends on the final value. The lower the final ionic strength gets, the faster is the self-assembly process. The variation of the salt composition at a given ionic strength has only a marginal effect, which depends on the ionic strength. The self-assembly obeys a step-growth process, where any intermediate cluster can coalesce with any other cluster. Interpretation of the data with a kinetic model based on the approach of von Smoluchowski follows a diffusion-limited cluster aggregation at ionic strength values lower than 30 mM. At an ionic strength of 30 mM, the model has to take into account a size dependence of the rate constant, and at 60 mM a transition is observed to a reaction-limited cluster aggregation.

Polyacrylates in the presence of an extraordinary monovalent cation-Solution behavior and metal nanoparticle formation.

Sodium polyacrylate (NaPA) in dilute aqueous solution at an ionic strength of [NaNO3] = 0.01M establishes a rich phase behavior in the presence of low amounts of silver cations, which were introduced at a few millimoles or less by replacing the corresponding amount of Na+ cations. Beyond an extremely low level of Ag+ cations, anionic PA chains aggregate. By increasing the concentration of Ag+, the aggregates become denser and keep on growing without limit. Once a certain range of [Ag+] is reached, the instantaneously formed dense aggregates remain stable. Irradiation of the PA aggregate solutions with UV-light induces formation of silver nanoparticles (Ag-Nps). Based on a combination of UV-vis spectroscopy, light scattering, transmission electron microscopy, and small angle neutron scattering, the mechanism of this NaPA assisted formation of Ag-Nps is studied. One focus of the study is lying on the effect of the two different solution states of dense aggregates, corresponding to the unstable growing AgPA aggregates and to the stable AgPA aggregates and another focus is aiming at the characterisation of the morphology of the generated hybrid particles composed of Ag-Nps and hosting PA chains.