The fibril formation of the neurodegenerative peptide amyloid β (Aβ42) is sensitive to solution conditions, and several proteins and peptides have been found to retard the process. Aβ42 fibril formation was followed with ThT fluorescence in the presence of polyamino acids (poly-glutamic acid, poly-lysine, and poly-threonine) and other polymers (poly(acrylic acid), poly(ethylenimine), and poly(diallyldimethylammonium chloride). An accelerating effect on the Aβ42 aggregation process is observed from all positively charged polymers, while no effect is seen from the negative or neutral polymers. The accelerating effect is dependent on the concentration of positive polymer in a highly reproducible manner. Acceleration is observed from a 1:500 polymer to Aβ42 weight ratio and up. Polyamino acids and the other polymers exert quantitatively the same effect at the same concentrations based on weight. Fibrils are formed in all cases as verified by transmission electron microscopy. The concentrations of polymers required for acceleration are too low to affect the Aβ42 aggregation process through increased ionic strength or molecular crowding effects. Instead, the acceleration seems to arise from the locally increased Aβ42 concentration near the polymers, which favors association and affects the electrostatic environment of the peptide.
The enzymatic activity of human carbonic anhydrase II (HCAII) was studied in the presence of nanoparticles of different nature and charge. Negatively charged nanoparticles inhibit HCAII whereas no effect is seen for positively charged particles. The kinetic effects were correlated with the strength of binding of the enzyme to the particle surface as measured by ITC and adsorption assays. Moreover, conformational changes upon adsorption were observed by circular dichroism. The main initial driving force for the adsorption of HCAII to nanoparticles is of electrostatic nature whereas the hydrophobic effect is not strong enough to drive the initial binding. This is corroborated by the fact that HCAII do not adsorb on positively charged hydrophobic polystyrene nanoparticles. Furthermore, the dehydration of the particle and protein surface seems to play an important role in the inactivation of HCAII by carboxyl-modified polystyrene nanoparticles. On the other hand, the inactivation by unmodified polystyrene nanoparticles is mainly driven by intramolecular interactions established between the protein and the nanoparticle surface upon conformational changes in the protein.
A kinetic study was carried out on various solvolytic reactions in water/NH4OT/isooctane microemulsions. The NH4OT surfactant is a derivative of the sodium salt of bis(2-ethylhexyl) sulfosuccinate (NaOT or AOT), where the Na+ counterion has been replaced by NH4+. The counterion substitution effects the phase diagram of the system, and therefore, NH4OT-based microemulsions with high water content reaching values of W = 350 (W = [H2O]/[NH4OT]) can be obtained. The presence of high W values suggests a transition in the microemulsion microstructure from water-in-oil (w/o) to oil-in-water (o/w), as was confirmed by conductivity and H-1 NMR self-diffusion measurements. The interpretation of the kinetic studies in terms of pseudophase formalism allows us to analyze the effect of the microemulsion on chemical reactivity, regardless of its microstructure. It has been confirmed that the values of the solvolytic rate constants at the interphase of oil-in-water microemulsions are similar to those obtained for aqueous SDS systems, showing that the hydration degree of the interphase of the oil-in-water microemulsions is independent of W. The influence of the surfactant counterion on the solvolytic rate constants was analyzed by comparing HOT-, NaOT-, and NH4OT-based microemulsions. An important influence on the rate constants caused by the changes in the structural properties of water has been observed as was confirmed by the water H-1 NMR signals.
The fibrillation process of the islet amyloid polypeptide (IAPP) and its fragment (IAPP(20-29)) was studied by means of Thioflavin T (ThT) fluorescence and transmission electron microscopy in the absence and presence of N-isopropylacrylamide:N-tert-butylacrylamide (NiPAM:BAM) copolymeric nanoparticles. The process was found to be strongly affected by the presence of the nanoparticles, which retard protein fibrillation as a function of the chemical surface properties of the nanoparticles. The NiPAM:BAM ratio was varied from 50:50 to 100:0. The nanoparticles with higher fraction of NiPAM imposed the strongest retardation of IAPP and IAPP(20-29) fibrillation. These particles have the strongest hydrogen bonding capacity due to the less bulky N-isopropyl group and thus less steric hindrance of the hydrogen-bonding groups of the nanoparticle polymer backbone. Kinetic fibrillation data, as monitored by ThT fluorescence and supported by surface plasmon resonance experiments, suggest that the peptide is strongly absorbed onto the surface of the nanoparticles. This interaction reduces the concentration of peptide free in solution available to proceed to fibrillation which results in an increased lag time of fibrillation, observed as a delayed onset of ThT fluorescence increase, plus a reduction of the amount of fibrils formed as indicated by the equilibrium values at the end of the fibrillation reaction. For the fragment (IAPP(20-29)), the presence of nanoparticles changes the mechanism of association from monomers to fibrils, by interfering with early oligomeric species along the fibrillation pathway.
Diffusion measurements by nuclear magnetic resonance (NMR) spectroscopy were used to investigate the host-guest association between beta-cyclodextrin (CD) and alkyltrimethylammonium bromide surfactants with different chain lengths, ranging from 6 up to 16 carbons. The scope and limitations of the method in the study of formation of inclusion complexes are discussed. The influences of the presence of CD in the micellization process have been studied, and the apparent critical micellar concentration and the self-diffusion coefficients of the species present in the systems have been calculated. The stoichiometries of the different complexes have been determined. Evidence for the formation of a 2:1 complex in the case Of C(16)TAB has been found.
Nanoparticles interfere with protein amyloid formation. Catalysis of the process may occur due to increased local protein concentration and nucleation on the nanoparticle surface, whereas tight binding or a large particle/protein surface area may lead to inhibition of protein aggregation. Here we show a clear correlation between the intrinsic protein stability and the nanoparticle effect on the aggregation rate. The results were reached for a series of five mutants of single-chain monellin differing in intrinsic stability toward denaturation, for which a correlation between protein stability and aggregation propensity has been previously documented by Szczepankiewicz et al. [Mol. Biosyst.20107 (2), 521-532]. The aggregation process was monitored by thioflavin T fluorescence in the absence and presence of copolymeric nanoparticles with different hydrophobic characters. For mutants with a high intrinsic stability and low intrinsic aggregation rate, we find that amyloid fibril formation is accelerated by nanoparticles. For mutants with a low intrinsic stability and high intrinsic aggregation rate, we find the opposite--a retardation of amyloid fibril formation by nanoparticles. Moreover, both catalytic and inhibitory effects are most pronounced with the least hydrophobic nanoparticles, which have a larger surface accessibility of hydrogen-bonding groups in the polymer backbone.
Casein (CN) micelles are naturally occurring colloidal protein aggregates present in a dispersed state in milk. In this paper we aim to obtain a detailed description of physicochemical properties of CN micelles over the entire size distribution using asymmetrical flow field-flow fractionation (AsFlFFF) connected to multiangle light scattering (MALS) and refractive index (RI) detection. Conclusions are drawn on the colloidal level regarding shape and conformation by comparison with models of colloidal particles. By using AsFlFFF-MALS-RI, it is concluded that the CN micelles are highly polydisperse with an average rms radius and hydrodynamic radius of 177 and 116 nm, respectively. The results show that the majority of CN micelles have a spherical shape, whereas a low concentration exists of larger and elongated aggregates. By comparison with models of aggregates of colloidal particles, the aggregates are shown to be anisotropic, e.g., aggregating linearly (threadlike) or in a sheet, rather than forming randomly spherical clusters. The results show that the characterization of colloidal dispersions with AsFlFFF-MALS-RI and the comparison with theoretical models are of a general character and, thus, of fundamental importance for colloidal dispersions.
The interaction between beta-cyclodextrin (CD) and gemini surfactant of the type alkyl-alpha,omega-bis(dodecyldimethyl-ammonium bromide) with different spacer lengths of 2, 8, and 10 carbons has been investigated by means of electric conductivity (EC) and proton self-diffusion NMR at 298 K. The formation of a 2: 1 (CD: gemini) complex in a two-step mechanism is observed with the first association constant (K-11) higher than the second one (K-21), but both relatively small in comparison with single C-12-tailed surfactant. The value of the association constants increased with spacer length both for the first and second associated CD, which indicates that the available space on the gemini molecule is important. The magnitudes of the association constant both for the first and second complexation are discussed. The first association constant is small ( when compared with the homologous single-chain surfactant) due to hydrophobic interaction between the hydrocarbon tails within the gemini molecule, while the second association constant shows no cooperativity and its magnitude is discussed in terms of steric constrains.