Chemical colloids versus biological colloids: a comparative study for the elucidation of the mechanism of protein fiber formation

Fiber formation from murine serum amyloid A1 (SAA) was compared to the linear aggregation and fiber formation of colloidal gold particles. Here we report the similarities of these processes. Upon incubation with acetic acid, SAA misfolds and adopts a new conformation, which we termed saa. saa apparently is less soluble than SAA in aqueous solution; it aggregates and forms nucleation units and then fibers. The fibers appear as a string of the nucleation units. Additionally, an external electric field promotes saa fiber formation. These properties of saa are reminiscent of colloidal gold formation from gold ions and one-dimensional aggregation of the gold colloids. Colloidal gold particles were also found to be capable of aggregating one-dimensionally under an electric field or in the presence of polylysine. These gold fibers resembled in structure that of saa fibers. In summary, protein aggregation and formation of fibers appear to follow the generalized principles derived in colloidal science for the aggregation of atoms and molecules, including polymers such as polypeptides. The analysis of colloidal gold formation and of one-dimensional aggregation provides a simple model system for the elucidation of some aspects of protein fiber formation.     

Charged polymers modulate retrovirus transduction via membrane charge neutralization and virus aggregation

The specific mechanisms of charged polymer modulation of retrovirus transduction were analyzed by characterizing their effects on virus transport and adsorption. From a standard colloidal perspective two mechanisms, charge shielding and virus aggregation, can potentially account for the experimentally observed changes in adsorption behavior and biophysical parameters due to charged polymers. Experimental testing revealed that both mechanisms could be at work depending on the characteristics of the cationic polymer. All cationic polymers enhanced adsorption and transduction via charge shielding; however, only polymers greater than 15 kDa in size were capable of enhancing these processes via the virus aggregation mechanism, explaining the higher efficiency enhancement of the high molecular weight molecules. The role of anionic polymers was also characterized and they were found to inhibit transduction via sequestration of cationic polymers, thereby preventing charge shielding and virus aggregation. Taken together, these findings suggest the basis for a revised physical model of virus transport that incorporates electrostatic interactions through both virus-cell repulsive and attractive interactions, as well as the aggregation state of the virus.     

Tau peptides and tau mutant protein aggregation inhibition by cationic polyethyleneimine and polyarginine

Tau protein plays a major role in Alzheimer's disease. The tau protein loses its functionality by self‐aggregation due to the two six‐amino acid sequences VQIVYK and VQIINK of the protein. Hence it is imperative to find therapeutics that could inhibit the self‐aggregation of this tau peptide fragments. Here, we study the inhibitory potential of a cationic polymer polyethyleneimine (PEI) and a cationic polypeptide arginine (Arg) on the aggregation of VQIVYK, and GKVQIINKLDL peptides, and tau mutant protein (P301L), found frequently in taupathy. Various characterization methods are employed including thioflavin S, transmission electron microscopy, and dynamic light scattering to study the aggregation/inhibition process in vitro. Results show that PEI and Arg significantly inhibit tau peptides and protein aggregation. The study could be applied to understand tau protein aggregation mechanism in the presence of cationic polymers.     

Crowding,Entropic Forces,and Confinement: Crucial Factors for Structures and Functions in the Cell Nucleus

The view of the cell nucleus as a crowded system of colloid particles and that chromosomes are giant self-avoiding polymers is stimulating rapid advances in our understanding of its structure and activities, thanks to concepts and experimental methods from colloid, polymer, soft matter, and nano sciences and to increased computational power for simulating macromolecules and polymers. This review summarizes current understanding of some characteristics of the molecular environment in the nucleus, of how intranuclear compartments are formed, and of how the genome is highly but precisely compacted, and underlines the crucial, subtle, and sometimes unintuitive effects on structures and reactions of entropic forces caused by the high concentration of macromolecules in the nucleus.     

Diffuse Reflectance Infrared Spectroscopic Identification of Dispersant/Particle Bonding Mechanisms in Functional Inks

In additive manufacturing, or 3D printing, material is deposited drop by drop, to create micron to macroscale layers. A typical inkjet ink is a colloidal dispersion containing approximately ten components including solvent, the nano to micron scale particles which will comprise the printed layer, polymeric dispersants to stabilize the particles, and polymers to tune layer strength, surface tension and viscosity. To rationally and efficiently formulate such an ink, it is crucial to know how the components interact. Specifically, which polymers bond to the particle surfaces and how are they attached? Answering this question requires an experimental procedure that discriminates between polymer adsorbed on the particles and free polymer. Further, the method must provide details about how the functional groups of the polymer interact with the particle. In this protocol, we show how to employ centrifugation to separate particles with adsorbed polymer from the rest of the ink, prepare the separated samples for spectroscopic measurement, and use Diffuse Reflectance Fourier Transform Infrared Spectroscopy (DRIFTS) for accurate determination of dispersant/particle bonding mechanisms. A significant advantage of this methodology is that it provides high level mechanistic detail using only simple, commonly available laboratory equipment. This makes crucial data available to almost any formulation laboratory. The method is most useful for inks composed of metal, ceramic, and metal oxide particles in the range of 100 nm or greater. Because of the density and particle size of these inks, they are readily separable with centrifugation. Further, the spectroscopic signatures of such particles are easy to distinguish from absorbed polymer. The primary limitation of this technique is that the spectroscopy is performed ex-situ on the separated and dried particles as opposed to the particles in dispersion. However, results from attenuated total reflectance spectra of the wet separated particles provide evidence for the validity of the DRIFTS measurement.     

Adsorption of gamma-globulin, a model protein for antibody, on colloidal particles

The effect of surface properties on the adsorption of bovine gamma-globulin, a model protein for antibody, was studied. Polystyrene latex (PS), hydrophilic copolymer lattices of styrene/2-hydroxyethyl methacrylate [P(S/HEMA)], styrene/ methacrylic acid [P(S/MAA)] and methyl methacrylate/ 2-hydroxyethyl methacrylate [P(MMA/HEMA)], and colloidal silica were used. The adsorption isotherms of gamma-globulin on these colloidal particles were measured as a function of pH and ionic strength. The hydrophilic particles showed low affinities for gamma-globulin at alkaline pH, while PS showed high affinities for gamma-globulin over the whole range of pH and ionic strength. The gamma-globulin adsorption on hydrophilic particles was highly reversible with respect to the pH and ionic strength compared with that on PS. These differences indicate that the dominant driving forces of adsorption are related to the hydrophilicity of particles. The adsorption isotherms of all colloidal particles showed the plateau values, and the order of maximum values of plateau adsorption was P(S/MAA) > PS or P(S/HEMA), silica > P(MMA/HEMA). Thus, they were also affected by the charged groups and the hydrophilicity of the surfaces. On the other hand, the plateau values of all colloidal particles were more or less symmetrical with a maximum at around the isoelectric point of gamma-globulin at an ionic strength of 0.01. This behavior is attributed to the important role of the lateral interaction between the adsorbed molecules at low ionic strength.     

On the temperature dependence of complex formation between chitosan and proteins

Chitosan is a biocompatible easily degradable polysaccharide, which, because of its positive charge, is able to interact favorably with deprotonated carboxyl groups of proteins. The strength of these charge-charge interactions is generally low, resulting in poor colloidal stability of the complexes. To investigate if other noncovalent forces contribute to stabilizing such systems, we have selected α-lactalbumin, β-lactoglobulin, β-casein, and human growth hormone, characterized by a common acidic pI value (～ 5) that ensures their overall negative charge at physiological pH. Binding energetics between chitosan and proteins was studied by isothermal titration calorimetry, whereas the thermal stability was assessed by differential scanning calorimetry. Our data show that colloidal stability of the particles depends on protein identity as well as temperature, indicating the involvement of nonelectrostatic interactions (e.g., hydrophobic effect) as driving forces for the complex formation. This suggests that chitosan-protein drug delivery systems can be improved through preparation process optimization with regard to temperature.     

Steric exclusion of cells. A mechanism of glycosaminoglycan-induced cell aggregation.

The mechanism by which chondroitin sulfate enhances both the self-aggregation and the concanavalin A (ConA)-induced agglutination of trypsin-dissociated embryonic chick retina cells was investigated. Studies with fluorescently labeled ConA showed no influence of chondroitin sulfate on patching or capping. When ³H- or ³⁵SO₄-labeled glycosaminoglycans or proteoglycans from retinas were added to freshly dissociated cells, an average of less than 2% of the label became associated with the unwashed cell pellet, and most of this was removed from the cells by a single wash. The presence of ConA did not alter the amount of binding by this criterion. Rapid cell aggregation in the absence of ConA was promoted by a number of natural and synthetic polymers. Aggregation rate bore a direct relationship to polymer viscosity at low viscosities and was inhibited at high viscosities, apparently due to reduced cell collision frequency. For any given polymer, aggregation was directly related to its molecular weight and concentration. Linear polymers were more effective than branched ones. Neutral polymers were as effective as those which were strongly polyanionic. Stable aggregates of formalin-fixed cells were not promoted by polymers. All of these observations are consistent with the hypothesis that enhancement of retina cell aggregation by physiological concentrations of glycosaminoglycans is due largely to steric exclusion of the cells by the polymer mesh. Although others have shown that glycosaminoglycans probably interact specifically with some cells, the evidence presented here suggests that these macromolecules by virtue of their excluded volumes could also have important non-specific influences on cell migration and cell reorganization during morphogenesis.     

Consensus features in amyloid fibrils: sheet-sheet recognition via a (polar or nonpolar) zipper structure

Amyloid fibrils characterized as highly intractable thread-like species are associated with many neurodegenerative diseases. Although neither the mechanism of amyloid formation nor the origin of amyloid toxicity is currently completely understood, the detailed three-dimensional atomic structures of the yeast protein Sup35 and Abeta amyloid protein determined by recent experiments provide the first and important step towards the comprehension of the pathogenesis and aggregation mechanisms of amyloid diseases. By analyzing these two amyloid peptides which have available crystal structures and other amyloid sequences with proposed structures using computational simulations, we delineate three common features in amyloid organizations and amyloid structures. These could contribute to an improved understanding of the molecular mechanism of amyloid formation, the nature of the aggregation driving forces that stabilize these structures and the development of potential therapeutic agents against amyloid diseases.     

Laser light scattering measurement of dextran-induced Streptococcus mutans aggregation.

Intensity fluctuation spectroscopy was used to study dextran-induced aggregation of Streptococcus mutans bacteria. Smoluchowski's theory of colloidal flocculation provided a consistent model of the agglutination process. Our experiments indicated that aggregation was inhibited by the negatively charged surfaces of the cells, while dextran polymers effectively bound organisms together. Our experimental data were consistent with the quantitative predictions of a polymer bridge model of agglutination.     

Stabilization of membranes upon interaction of amphipathic polymers with membrane proteins

Amphipathic polymers derived from polysaccharides, namely hydrophobically modified pullulans, were previously suggested to be useful as polymeric substitutes of ordinary surfactants for efficient and structure-conserving solubilization of membrane proteins, and one such polymer, 18C(10), was optimized for solubilization of proteins derived from bacterial outer membranes (Duval-Terrie et al. 2003). We asked whether a similar ability to solubilize proteins could also be demonstrated in eukaryotic membranes, namely sarcoplasmic reticulum (SR) fragments, the major protein of which is SERCA1a, an integral membrane protein with Ca(2+)-dependent ATPase and Ca(2+)-pumping activity. We found that 18C(10)-mediated solubilization of these SR membranes did not occur. Simultaneously, however, we found that low amounts of this hydrophobically modified pullulan were very efficient at preventing long-term aggregation of these SR membranes. This presumably occurred because the negatively charged polymer coated the membranous vesicles with a hydrophilic corona (a property shared by many other amphipathic polymers), and thus minimized their flocculation. Reminiscent of the old Arabic gum, which stabilizes Indian ink by coating charcoal particles, the newly designed amphipathic polymers might therefore unintentionally prove useful also for stabilization of membrane suspensions.     

A review of the potential and versatility of colloidal gold cytochemical labeling for molecular morphology.

In the present article we review several postembedding cytochemical techniques using the colloidal gold marker. Owing to the high atomic number of gold, the colloidal gold particles are electron dense. They are spherical in shape and can be prepared in sizes from 1 to 25 nm, which renders this marker among the best for electron microscopy. In addition, because it can be bound to several molecules, this marker has the advantage of being extremely versatile. Combined to immunoglobulins or immunoglobulin-binding proteins (protein A), it has been applied successfully in immunocytochemistry. Colloidal gold particles 5-15 nm in size are excellent for postembedding cytochemistry. Particles of smaller size, such as 1 nm, must be silver enhanced to be visualized by transmission electron microscopy. We have elected to review the superiority of indirect immunocytochemical approaches using IgG-gold or protein A-gold (protein G-gold and protein AG-gold). Lectins or enzymes can be tagged with colloidal gold particles, and the corresponding lectin-gold and enzyme-gold techniques have specific advantages and great potential. Using an indirect digoxigenin-tagged nucleotide and an antidigoxigenin probe, colloidal gold technology can also be used for in situ hybridization at the electron microscope level. Affinity characteristics lie behind all cytochemical techniques and several molecules displaying high affinity properties can also be beneficial for colloidal gold electron microscopy cytochemistry. All of these techniques can be combined in various ways to produce multiple labelings of several binding sites on the same tissue section. Colloidal gold is particulate and can easily be counted; thus the cytochemical signal can be evaluated quantitatively, introducing further advantages to the use of the colloidal gold marker. Finally, several combinations and multiple step procedures have been designed to amplify the final signal which renders the techniques more sensitive. The approaches reviewed here have been applied successfully in different fields of cell and molecular biology, cell pathology, plant biology and pathology, microbiology and virology. The potential of the approaches is emphasized in addition to different ways to assess specificity, sensitivity and accuracy of results.     

A Review of the Potential and Versatility of Colloidal Gold Cytochemical Labeling for Molecular Morphology

In the present article we review several postembedding cytochemical techniques using the colloidal gold marker. Owing to the high atomic number of gold, the colloidal gold particles are electron dense. They are spherical in shape and can be prepared in sizes from 1 to 25 nm, which renders this marker among the best for electron microscopy. In addition, because it can be bound to several molecules, this marker has the advantage of being extremely versatile. Combined to immunoglobulins or immunoglobulin-binding proteins (protein A), it has been applied successfully in immunocytochemistry. Colloidal gold particles 5–15 nm in size are excellent for postembedding cytochemistry. Particles of smaller size, such as 1 nm, must be silver enhanced to be visualized by transmission electron microscopy. We have elected to review the superiority of indirect immunocytochemical approaches using IgG-gold or protein A-gold (protein G-gold and protein AG-gold). Lectins or enzymes can be tagged with colloidal gold particles, and the corresponding lectin-gold and enzyme-gold techniques have specific advantages and great potential. Using an indirect digoxigenin-tagged nucleotide and an antidigoxigenin probe, colloidal gold technology can also be used for in situ hybridization at the electron microscope level. Affinity characteristics lie behind all cytochemical techniques and several molecules displaying high affinity properties can also be beneficial for colloidal gold electron microscopy cytochemistry. All of these techniques can be combined in various ways to produce multiple labelings of several binding sites on the same tissue section. Colloidal gold is particulate and can easily be counted; thus the cytochemical signal can be evaluated quantitatively, introducing further advantages to the use of the colloidal gold marker. Finally, several combinations and multiple step procedures have been designed to amplify the final signal which renders the techniques more sensitive. The approaches reviewed here have been applied successfully in different fields of cell and molecular biology, cell pathology, plant biology and pathology, microbiology and virology. The potential of the approaches is emphasized in addition to different ways to assess specificity, sensitivity and accuracy of results.     

Insight into the correlation between lag time and aggregation rate in the kinetics of protein aggregation

Under favorable conditions, many proteins can assemble into macroscopically large aggregates such as the amyloid fibrils that are associated with Alzheimer's, Parkinson's, and other neurological and systemic diseases. The overall process of protein aggregation is characterized by initial lag time during which no detectable aggregation occurs in the solution and by maximal aggregation rate at which the dissolved protein converts into aggregates. In this study, the correlation between the lag time and the maximal rate of protein aggregation is analyzed. It is found that the product of these two quantities depends on a single numerical parameter, the kinetic index of the curve quantifying the time evolution of the fraction of protein aggregated. As this index depends relatively little on the conditions and/or system studied, our finding provides insight into why for many experiments the values of the product of the lag time and the maximal aggregation rate are often equal or quite close to each other. It is shown how the kinetic index is related to a basic kinetic parameter of a recently proposed theory of protein aggregation. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Effects of dextran molecular weight on red blood cell aggregation

The reversible aggregation of human red blood cells (RBC) by proteins or polymers continues to be of biologic and biophysical interest, yet the mechanistic details governing the process are still being explored. Although a depletion model with osmotic attractive forces due to polymer depletion near the RBC surface has been proposed for aggregation by the neutral polyglucose dextran, its applicability at high molecular mass has not been established. In this study, RBC aggregation was measured over a wide range of dextran molecular mass (70 kDa to 28 MDa) at concentrations ≤2 g/dL. Our results indicate that aggregation does not monotonically increase with polymer size; instead, it demonstrates an optimum dextran molecular mass around 200-500 kDa. We used a model for depletion-mediated RBC aggregation to calculate the expected depletion energies. This model was found to be consistent with the experimental results and thus provides new insight into polymer-RBC interactions.     

Aggregation of phospholipid vesicles by water-soluble polymers.

Water-soluble polymers such as dextran and polyethylene glycol are known to induce aggregation and size growth of phospholipid vesicles. The present study addresses the dependence of these processes on vesicle size and concentration, polymer molecular weight, temperature, and compartmentalization of the vesicles and polymers, using static and dynamic light scattering. Increasing the molecular weight of the polymers resulted in a reduction of the concentration of polymer needed for induction of aggregation of small unilamellar vesicles. The aggregation was fully reversible (by dilution), within a few seconds, up to a polymer concentration of at least 20 wt %. At relatively low phosphatidylcholine (PC) concentrations (up to approximately 1 mM), increasing the PC concentration resulted in faster kinetics of aggregation and reduced the threshold concentration of polymer required for rapid aggregation (CA). At higher PC concentrations, CA was only slightly dependent on the concentration of PC and was approximately equal to the overlapping concentration of the polymer (C*). The extent of aggregation was similar at 37 and 4 degrees C. Aggregation of large unilamellar vesicles required a lower polymer concentration, probably because aggregation occurs in a secondary minimum (without surface contact). In contrast to experiments in which the polymers were added directly to the vesicles, dialysis of the vesicles against polymer-containing solutions did not induce aggregation. Based on this result, it appears that exclusion of polymer from the hydration sphere of vesicles and the consequent depletion of polymer molecules from clusters of aggregated vesicles play the central role in the induction of reversible vesicle aggregation. The results of all the other experiments are consistent with this conclusion.     

Stabilization of proteins encapsulated in injectable poly (lactide- co-glycolide)

Controlled release from biodegradable polymers is a novel approach to replace daily painful injections of protein drugs. A major obstacle to development of these polymers is the need to retain the structure and biological activity of encapsulated proteins during months of incubation under physiological conditions. We encapsulated bovine serum albumin (BSA) in injectable poly(DL-lactide- co-glycolide) (PLGA) 50/50 cylindrical implants and determined the mechanism of BSA instability. Simulations of the polymer microclimate revealed that moisture and acidic pH (<3) triggered unfolding of encapsulated BSA, resulting in peptide bond hydrolysis and noncovalent aggregation. To neutralize the acids liberated by the biodegradable lactic/glycolic acid-based polyester, we coincorporated into the polymer an antacid, Mg(OH)2, which increased microclimate pH and prevented BSA structural losses and aggregation for over one month. We successfully applied this stabilization approach in both cylinder- and microsphere-injectable configurations and for delivery of angiogenic basic fibroblast growth factor and bone-regenerating bone morphogenetic protein-2.     

Lateral forces acting between particles in liquid films or lipid membranes

To investigate the mechanism of formation of 2D arrays of protein macromolecules in liquid films we carried out model experiments with μm-sized latex particles. The direct observations revealed that the process of ordering is triggered by attractive lateral capillary forces due to the overlap of the menisci formed around the particles. Two types of lateral capillary forces, flotation and immersion, can be distinguished, and a theory of these interactions is developed. Similar forces are operative between inclusions (proteins) incorporated in lipid membranes. We develop an appropriate model of a lipid bilayer, which is described as an elastic layer (the hydrocarbon chain region) sandwiched between two Gibbs dividing surfaces (the two headgroup regions). The range of the interaction between two cylindrical inclusions turns out to be of the order of several inclusion radii. The results, which are in qualitative agreement with the experimental observations, can be applied to the interpretation of membrane processes and mechanisms such as protein aggregation in lipid membranes.     

Surface interaction forces of cellulose nanocrystals grafted with thermoresponsive polymer brushes

The colloidal stability and thermoresponsive behavior of poly(N-isopropylacrylamide) brushes grafted from cellulose nanocrystals (CNCs) of varying graft densities and molecular weights was investigated. Indication of the grafted polymer brushes was obtained after AFM imaging of CNCs adsorbed on silica. Also, aggregation of the nanoparticles carrying grafts of high degree of polymerization was observed. The responsiveness of grafted CNCs in aqueous dispersions and as an ultrathin film was evaluated by using light scattering, viscosimetry, and colloidal probe microscopy (CPM). Light transmittance measurements showed temperature-dependent aggregation originating from the different graft densities and molecular weights. The lower critical solution temperature (LCST) of grafted poly(NiPAAm) brushes was found to decrease with the ionic strength, as is the case for free poly(NiPAAm) in aqueous solution. Thermal responsive behavior of grafted CNCs in aqueous dispersions was observed by a sharp increase in dispersion viscosity as the temperature approached the LCST. CPM in liquid media for asymmetric systems consisting of ultrathin films of CNCs and a colloidal silica probe showed the distinctive effects of the grafted polymer brushes on interaction and adhesive forces. The origin of such forces was found to be mainly electrostatic and steric in the case of bare and grafted CNCs, respectively. A decrease in the onset of attractive and adhesion forces of grafted CNCs films were observed with the ionic strength of the aqueous solution. The decreased mobility of polymer brushes upon partial collapse and decreased availability of hydrogen bonding sites with higher electrolyte concentration were hypothesized as the main reasons for the less prominent polymer bridging between interacting surfaces.