Thermoreversible gelation in aqueous binary solvents of chemically modified agarose

The thermoreversible gelation of chemically modified agarose has been studied in aqueous binary solvents (dimethyl sulfoxide and a series of formamide) by differential calorimetry, mechanical testing, and small-angle neutron scattering. The temperature–composition phase diagrams have been established. It is concluded that gelation is promoted by the formation of ternary complexes modified agarose/water/cosolvent, wherein the cosolvent mediates the interaction between chains through the formation of electrostatic interactions.     

Effects of salt concentration on association of the amyloid protofilaments of hen egg white lysozyme studied by time-resolved neutron scattering

Various proteins have been shown to form various aggregated structures including the filamentous aggregates known as amyloid fibrils depending on the solution conditions. Hen egg white lysozyme (HEWL) is one of the proteins that form the amyloid fibrils. To gain insight into the mechanism of this polymorphism of the aggregated structures, we employed a model system consisting of HEWL, pure water, and ethanol, and investigated the kinetic process of the fibril formation in various salt concentrations with time-resolved neutron scattering. It was shown that by addition of NaCl in a range between 0.3 mM and 1.0 mM to HEWL solution in 90% ethanol, gelation occurred, and this gelation proceeded through a two-step process: the lateral association of the protofilaments, followed by the cross-linking of these fibrils formed. Both the structures of the fibrils and the rate of the gelation depended on NaCl concentration. The average structures of the fibrils formed at 1.0 mM NaCl were characterized by the radius of gyration of their cross-section (45.9(+/-0.4)A) and the number of the protofilaments within the fibril (4.10(+/-0.12)), corresponding to the mature amyloid fibrils. A range of intermediate structures was formed below 1 mM NaCl. Above 2 mM NaCl, precipitation occurred because of the formation of amorphous aggregates. Here the branch point to the formation of the mature amyloid fibrils or to the amorphous aggregates was after the formation of the protofilaments. Sensitivity of the aggregated structures to salt concentration suggests that electrostatic interaction plays an essential role in the formation of these structures. The structural diversity both in the fibrils and the aggregated structures of the fibrils can be interpreted in terms of the difference in the degree of the electrostatic shielding at different salt concentrations.     

Folding and dimerization of the ionic peptide EAK 16-IV

Folding and dimerization of an ionic polyalanine-based peptide chain (EAK16-IV) are simulated with nonspecific interactions. It is found that there is a competition between two kinds of structural motifs under different strengths of electrostatic interactions. The dominance of hairpin-like structures would be realized with a strong electrostatic interaction both thermodynamically and kinetically, showing the importance of the electrostatic interaction on the formation of hairpin-like structures. Simulations on the dimerization with strong electrostatic interaction are also carried out. It is found that the concentration contributes essentially to the shape of the dimers. These studies demonstrate that the strong interactions and kinetic factors might be important for the ordered amyloid aggregates.     

Transient tropoelastin nanoparticles are early-stage intermediates in the coacervation of human tropoelastin whose aggregation is facilitated by heparan sulfate and heparin decasaccharides

Tropoelastin assembly is a key step in the formation of elastin. We consider how nanoscale intracellular assemblies of tropoelastin can congregate in an extracellular environment to give microscale aggregates. We describe novel 200–300 nm spherical particles that serve as intermediates in the formation of the coacervate. Their aggregation gives 800 nm to 1 µm species. This process is facilitated by heparan sulfate and dermatan sulfate interactions which effectively lower the critical concentration to facilitate this transition. This coacervation process was examined using a panel of heparin chains of various lengths and showed greatest efficacy for the decasaccharide, followed by the octasaccharide, while the hexasaccharide displayed the shortest efficacious length. We propose that these oligosaccharide interactions enable the charge-mediated aggregation of positively charged tropoelastin. This biochemistry models glycosaminoglycan interactions on the cell surface during elastogenesis which is characterized by the clustering of nascent tropoelastin aggregates to form micron-sized spherules.     

Raman study of the interaction between polyamines and a GC oligonucleotide.

The interaction between the oligonucleotide d[G(CG)(7)]. d[C(GC)(7)] and the three biogenic polyamines putrescine, spermidine, and spermine under physiological conditions has been studied by Raman spectroscopy. The results indicate the formation of highly ordered aggregated structures in solution, largely stabilized by electrostatic attractions, which have been described as cholesteric phases. Aggregation seems to be preceded by a partial B --> Z conformational transition for spermidine and spermine, which would allow for a deeper oligonucleotide-polyamine interaction. Interaction with the nucleic bases has also been evidenced for aggregates. At low polyamine concentrations the preferential binding sites are similar to those proposed for their interactions with ct-DNA. With increasing the polyamine concentration, the oligonucleotide-polyamine interactions involve both minor and major grooves, which is consistent with the formation of cholesteric phases.     

Self-assembly of repeat proteins: Concepts and design of new interfaces

In nature, assembled protein structures offer the most complex functional structures. The understanding of the mechanisms ruling protein–protein interactions opens the door to manipulate protein assemblies in a rational way. Proteins are versatile scaffolds with great potential as tools in nanotechnology and biomedicine because of their chemical, structural, and functional versatility. Currently, bottom-up self-assembly based on biomolecular interactions of small and well-defined components, is an attractive approach to biomolecular engineering and biomaterial design. Specifically, repeat proteins are simplified systems for this purpose.In this work, we provide an overview of fundamental concepts of the design of new protein interfaces. We describe an experimental approach to form higher order architectures by a bottom-up assembly of repeated building blocks. For this purpose, we use designed consensus tetratricopeptide repeat proteins (CTPRs). CTPR arrays contain multiple identical repeats that interact through a single inter-repeat interface to form elongated superhelices. Introducing a novel interface along the CTPR superhelix allows two CTPR molecules to assemble into protein nanotubes. We apply three approaches to form protein nanotubes: electrostatic interactions, hydrophobic interactions, and π-π interactions. We isolate and characterize the stability and shape of the formed dimers and analyze the nanotube formation considering the energy of the interaction and the structure in the three different models. These studies provide insights into the design of novel protein interfaces for the control of the assembly into more complex structures, which will open the door to the rational design of nanostructures and ordered materials for many potential applications in nanotechnology.     

Investigation at residue level of the early steps during the assembly of two proteins into supramolecular objects

Understanding the driving forces governing protein assembly requires the characterization of interactions at molecular level. We focus on two homologous oppositely charged proteins, lysozyme and α-lactalbumin, which can assemble into microspheres. The assembly early steps were characterized through the identification of interacting surfaces monitored at residue level by NMR chemical shift perturbations by titrating one (15)N-labeled protein with its unlabeled partner. While α-lactalbumin has a narrow interacting site, lysozyme has interacting sites scattered on a broad surface. The further assembly of these rather unspecific heterodimers into tetramers leads to the establishment of well-defined interaction sites. Within the tetramers, most of the electrostatic charge patches on the protein surfaces are shielded. Then, hydrophobic interactions, which are possible because α-lactalbumin is in a partially folded state, become preponderant, leading to the formation of larger oligomers. This approach will be particularly useful for rationalizing the design of protein assemblies as nanoscale devices.     

Oligomerization and phase transitions in aqueous solutions of native and truncated human beta B1-crystallin

Human betaB1-crystallin is a major eye-lens protein that undergoes in vivo truncation at the N-terminus with aging. By studying native betaB1 and truncated betaB1DeltaN41, which mimics an age-related in vivo truncation, we have determined quantitatively the effect of truncation on the oligomerization and phase transition properties of betaB1 aqueous solutions. The oligomerization studies show that the energy of attraction between the betaB1DeltaN41 proteins is about 10% greater than that of the betaB1 proteins. We have found that betaB1DeltaN41 aqueous solutions undergo two distinct types of phase transitions. The first phase transition involves an initial formation of thin rodlike assemblies, which then evolve to form crystals. The induction time for the formation of rodlike assemblies is sensitive to oligomerization. The second phase transition can be described as liquid-liquid phase separation (LLPS) accompanied by gelation within the protein-rich phase. We refer to this process as heterogeneous gelation. These two phase transitions are not observed in the case of betaB1 aqueous solutions. However, upon the addition of poly(ethylene glycol) (PEG), we observe heterogeneous gelation also for betaB1. Our PEG experiments allow us to estimate the difference in phase separation temperatures between betaB1 and betaB1DeltaN41. This difference is consistent with the increase in energy of attraction found in our oligomerization studies. Our work suggests that truncation is a cataractogenic modification since it favors protein condensation and the consequent formation of light scattering elements, and highlights the importance of the N-terminus of betaB1 in maintaining lens transparency.     

Phase Transition and Optical Properties of DNA–Gold Nanoparticle Assemblies

We review recent work on DNA-linked gold nanoparticle assemblies. The synthesis, properties, and phase behavior of such DNA–gold nanoparticle assemblies are described. These nanoparticle assemblies have strong optical extinction in the ultraviolet and visible light regions; hence, the technique is used to study the kinetics and phase transitions of DNA–gold nanoparticle assemblies. The melting transition of DNA–gold nanoparticle assemblies shows unusual trends compared to those of free DNA. The phase transitions are influenced by many parameters, such as nanoparticle size, DNA sequence, DNA grafting density, DNA linker length, interparticle distance, base pairing defects, and disorders. The physics of the DNA–gold nanoparticle assemblies can be understood in terms of the phase behavior of complex fluids, with the colloidal gold interaction potential dominated by DNA hybridization energies.     

Modulation of assembly of TDP-43 low-complexity domain by heparin: From droplets to amyloid fibrils

TAR DNA-binding protein 43 (TDP-43) is an RNA-regulating protein that carries out many cellular functions through liquid-liquid phase separation (LLPS). The LLPS of TDP-43 is mediated by its C-terminal low-complexity domain (TDP43-LCD) corresponding to the region 267–414. In neurodegenerative disorders amyotrophic lateral sclerosis and frontotemporal dementia, pathological inclusions of the TDP-43 are found that are rich in the C-terminal fragments of ～25 and ～35 kDa, of which TDP43-LCD is a part. Thus, understanding the assembly process of TDP43-LCD is essential, given its involvement in the formation of both functional liquid-like assemblies and solid- or gel-like pathological aggregates. Here, we show that the solution pH and salt modulate TDP43-LCD LLPS. A gradual reduction in the pH below its isoelectric point of 9.8 results in a monotonic decrease of TDP43-LCD LLPS due to charge-charge repulsion between monomers, while at pH 6 and below no LLPS was observed. The addition of heparin to TDP43-LCD solution at pH 6, at a 1:2 heparin-to-TDP43-LCD molar ratio, promotes TDP43-LCD LLPS, while at higher concentration, it disrupts LLPS through a reentrant phase transition. Upon incubation at pH 6, TDP43-LCD undergoes gelation without phase separation. However, in the reentrant regime in the presence of a high heparin concentration, it forms thick amyloid aggregates that are significantly more SDS resistant than the gel. The results indicate that the material nature of the TDP43-LCD assembly products can be modulated by heparin which is significant in the context of liquid-to-solid phase transition observed in TDP-43 proteinopathies. Our findings are also crucial in relation to similar transitions that could occur due to alteration in the molecular level interactions among various multivalent biomolecules involving other LCDs and RNAs.     

Cold-Set Whey Protein Gels with Addition of Polysaccharides

Cold-set whey protein (WP) gels with addition of xanthan or guar were evaluated by mechanical properties and scanning electron microscopy. Gels were formed after the addition of different amounts of glucono-δ-lactone to thermally denatured WP solutions, leading to different acidification rates and final pH values. At lower acidification rates and higher final pH, gels showed more discontinuous structure and weaker and less elastic network, which was attributed to a predominance of phase separation during gel formation due to slower gelation kinetics. In contrast, at higher acidification rates and lower final pHs, gelation prevailed over phase separation, favoring the formation of less porous structures, resulting in stronger and more elastic gels. The gels’ fractal dimension (D _f; structure complexity) and lacunarity were also influenced by the simultaneous effects of gelation and phase separation. For systems where phase separation was the prevailing mechanism, greater lacunarity parameters were usually observed, describing the heterogeneity of pore distribution, while the opposite occurred at prevailing gelation conditions. Increase in guar concentration or lower final pH of xanthan gels entailed in D _f reduction, while the increase in xanthan concentration resulted in higher D _f. Such a result suggests that the network contour length was rugged, but this pattern was reduced by the increase of electrostatic interactions among WP and xanthan. Guar addition caused the formation of gel network with smoother surfaces, which could be attributed to the guar–protein excluded volume effects leading to an increase in protein–protein interactions.     

Trivaline Molecular Complexes with Trinucleotides Form in Solution the Extended,about 1000 Å in Length Structures

Abstract

The fluorescence, flow linear dichroism and electron microscopy (EM) have shown the trivaline ability to interact in solution with certain molecules of trinucleotides. This interaction results in formation of extended structures up to several. thousand angstroms in length. Such structures were observed for trivaline complexes with homopurine, homopyrimidine or random sequences of deoxyribo- and ribonucleotides, independently of the presence or absence of the terminal 5′-phosphate residue. A model of such a structural organization is proposed. An elementary structural unit consists of a trivaline β-dimer and adsorbed trinucleotide. So, “dimeric” complex is formed. Two such “dimeric” complexes combine with each other by means of peptide-peptide contacts (as with β-sandwich). So, “tetrameric” complex is formed It has a dyad axis. Two such structural units combine with each other by means of Hoogsteen's hydrogen bonds. So, “octamreic” complex is formed. It has three mutually perpendicular dyad axes. The “octameric” complexes appear to be able to combine with each other by means of stacking interactions, and to form the regular organized aggregates consisting of many dozens of elementary units. So, “stacking” structure is formed. The “octameric” complex is the symmetry translational unit of such a stucture. The spatial position of the bases in all these structures is additionally fixed by the nucleo-peptide interactions. These aggregates have the appearance of extended structures on electron micrographs.     

Gelation by phase separation in a whey protein system: in-situ kinetics of aggregation

The aggregation and gelation properties of beta-lactoglobulin (BLG), a globular protein from milk, was studied in aqueous ethanol solutions at room temperature. The phase state diagrams as a function of pH and ethanol concentration showed that a gel structure appeared after a period ranging from 1 min to 1 week, depending on the physico-chemical conditions. The in-situ kinetics of aggregation were followed by several methods in order to obtain a better understanding of the building of aggregates by the addition of ethanol. It was shown that the aggregation kinetics highly depended upon the pH, the process being fastest at pH 7. Viscoelasticity and infrared measurements indicated that alcohol-induced gelation would proceed via a two-step mechanism: small aggregates loosely connected between them were first built up; a real network took place in a second step. The coarse and irregular structures formed in aqueous ethanol gels revealed by confocal laser scanning microscopy could be analysed in terms of a phase separation. This observation was supported by a syneresis phenomenon visible in the final gel state. BLG in water-ethanol solution would undergo either an inhibition of the demixing by gelation or a binary phase separation accompanied by an irreversible gelation transition.     

Calorimetric investigation of polymyxin binding to phosphatidic acid bilayers

The cooperative binding process between the antibiotic peptide polymyxin-B and negatively-charged phosphatidic acid bilayers was investigated by differential thermal analysis and completed by fluorescence polarization measurements. The sigmoidal binding curves were analyzed in terms of the interaction energy within a domain formed by polymyxin and phosphatidic acid molecules. The formation of such a heterogeneous domain structure was favoured by high concentration of external monovalent ions. The cooperativity of the binding increased while a charge-induced decrease in the phase transition temperature of the pure lipid phase was observed with increasing ion concentration at a given pH. The reduced lateral coupling within the lipid bilayer in the presence of salt ions, as demonstrated by an increase in the lipid phase transition enthalpy, was considered to facilitate the cooperative domain formation. Moreover, an increase in the cooperativity of the polymyxin binding could be observed if phosphatidic acids of smaller chain length and thus of a lowered phase transition temperature were used. By the use of chemically-modified polymyxin we were able to demonstrate the effect of electrostatic and hydrophobic interaction. Acetylated polymyxin with a reduced positive charge was used to demonstrate the pure hydrophobic effect of polymyxin binding leading to a decrease in the phosphatidic acid phase transition temperature by about 20°C. The cooperativity of the binding was strongly reduced. Cleavage of the hydrophobic polymyxin tail yielded a colistinnonapeptide which caused an electrostatically-induced increase in the phosphatidic acid phase transition temperature. With unmodified polymyxin we observed the combined effects of electrostatic as well as hydrophobic interaction making this model system interesting for the understanding of lipid-protein interactions. Evidence is presented that the formation of the polymyxin-phosphatidic acid complex is a lateral phase separation phenomenon.     

Resolving self-association of a therapeutic antibody by formulation optimization and molecular approaches

A common challenge encountered during development of high concentration monoclonal antibody formulations is preventing self-association. Depending on the antibody and its formulation, self-association can be seen as aggregation, precipitation, opalescence or phase separation. Here we report on an unusual manifestation of self-association, formation of a semi-solid gel or “gelation." Therapeutic monoclonal antibody C4 was isolated from human B cells based on its strong potency in neutralizing bacterial toxin in animal models. The purified antibody possessed the unusual property of forming a firm, opaque white gel when it was formulated at concentrations >30 mg/mL and the temperature was <6°C. Gel formation was reversible with temperature. Gelation was affected by salt concentration or pH, suggesting an electrostatic interaction between IgG monomers. A comparison of the C4 amino acid sequences to consensus germline sequences revealed differences in framework regions. A C4 variant in which the framework sequence was restored to the consensus germline sequence did not gel at 100 mg/mL at temperatures as low as 1°C. Additional genetic analysis was used to predict the key residue(s) involved in the gelation. Strikingly, a single substitution in the native antibody, replacing heavy chain glutamate 23 with lysine (E23K), was sufficient to prevent gelation. These results indicate that the framework region is involved in intermolecular interactions. The temperature dependence of gelation may be related to conformational changes near glutamate 23 or the regions it interacts with. Molecular engineering of the framework can be an effective approach to resolve the solubility issues of therapeutic antibodies.     

Self Assembly of Short Aromatic Peptides into Amyloid Fibrils and Related Nanostructures

The formation of amyloid fibrils is the hallmark of more than twenty human disorders of unrelated etiology. In all these cases, ordered fibrillar protein assemblies with a diameter of 7–10 nm are being observed. In spite of the great clinical important of amyloidassociated diseases, the molecular recognition and self-assembly processes that lead to the formation of the fibrils are not fully understood. One direction to decipher the mechanism of amyloid formation is the use of short peptides fragments as model systems. Short peptide fragments, as short as pentapeptides, were shown to form typical amyloid assemblies in vitro that have ultrastructural, biophysical, and cytotoxic properties, as those of assemblies that are being formed by full length polypeptides. When we analyzed such short fragments, we identified the central role of aromatic moieties in the ability to aggregate into ordered nano-fibrillar structures. This notion allowed us to discover additional very short amyloidogenic peptides as well as other aromatic peptide motifs, which can form various assemblies at the nano-scale (including nanotubes, nanospheres, and macroscopic hydrogels with nano-scale order). Other practical utilization of this concept, together with novel β breakage methods, is their use for the development of novel classes of amyloid formation inhibitors.Key Words: Alzheimer''s disease, amyloid disease, molecular recognition, nanostructures, protein aggregation, protein misfolding, self-assembly, type II diabetes     

Structural investigation of beta-lactoglobulin gelation in ethanol/water solutions.

The aggregation and gelation properties of beta-lactoglobulin (BLG), a globular protein from milk, was studied in hydro-ethanolic solutions (50/50% (v/v)) at room temperature. The phase state diagrams as a function of pH and ethanol concentration showed that a gel structure appeared after a period ranging from 1 min to 1 week depending on the physico-chemical conditions. The aggregation kinetics, studied by infrared spectroscopy and dynamical rheological measurements, highly depended upon the pH; the process being the fastest at pH 7. Alcohol-induced aggregation of BLG was characterized by the formation of intermolecular hydrogen bonded beta-sheet structures. Small angle neutron scattering indicated that the aggregates structures in the final gels were similar at pH 7, 8 and 9. Through the data obtained at the molecular and macroscopic levels, it can be concluded that the kinetics of gelation were pH dependent while the spatial arrangements of the aggregates were similar in the final structures. The heterogeneous structures formed in hydro-ethanolic gels could be analysed in terms of a phase separation, the syneresis being the final visible state.     

Calorimetric investigation of polymyxin binding to phosphatidic acid bilayers

The cooperative binding process between the antibiotic peptide polymyxin-B and negatively-charged phosphatidic acid bilayers was investigated by differential thermal analysis and completed by fluorescence polarization measurements. The sigmoidal binding curves were analyzed in terms of the interaction energy within a domain formed by polymyxin and phosphatidic acid molecules. The formation of such a heterogeneous domain structure was favoured by high concentration of external monovalent ions. The cooperativity of the binding increased while a charge-induced decrease in the phase transition temperature of the pure lipid phase was observed with increasing ion concentration at a given pH. The reduced lateral coupling within the lipid bilayer in the presence of salt ions, as demonstrated by an increase in the lipid phase transition enthalpy, was considered to facilitate the cooperative domain formation. Moreover, an increase in the cooperativity of the polymyxin binding could be observed if phosphatidic acids of smaller chain length and thus of a lowered phase transition temperature were used. By the use of chemically-modified polymyxin we were able to demonstrate the effect of electrostatic and hydrophobic interaction. Acetylated polymyxin with a reduced positive charge was used to demonstrate the pure hydrophobic effect of polymyxin binding leading to a decrease in the phosphatidic acid phase transition temperature by about 20 degrees C. The cooperativity of the binding was strongly reduced. Cleavage of the hydrophobic polymyxin tail yielded a colistinnonapeptide which caused an electrostatically-induced increase in the phosphatidic acid phase transition temperature. With unmodified polymyxin we observed the combined effects of electrostatic as well as hydrophobic interaction making this model system interesting for the understanding of lipid-protein interactions. Evidence is presented that the formation of the polymyxin-phosphatidic acid complex is a lateral phase separation phenomenon.