The critical micellar concentration (cmc) and the demicellization enthalpy ΔH demic of the primary aggregates of sodium cholate (NaC) and sodium deoxycholate (NaDC) in water and 0.1 M NaCl at pH 7.5 were determined by isothermal titration calorimetry (ITC). The cmc of NaC and NaDC in water and 0.1 M NaCl at pH 7.5 shows a minimum between 295 and 300 K. With increasing ionic strength, the cmc of the bile salts decreases. ΔH demic is strongly temperature-dependent but shows almost no dependence on the ionic strength. For comparison with other systems, the thermodynamic parameters ΔG demic and ΔS demic associated with the demicellization process were calculated using the pseudo-phase-separation model. From the temperature dependence of ΔH demic, the change in heat capacity ΔCp demic for the demicellization process was determined. The data obtained for ΔCp demic are positive and at 298 K have values of 250 J·mol-1·K-1 for NaC and 350 J·mol-1·K-1 for NaDC. These values correspond to changes in the exposed hydrophobic surface area of 1.1−1.5 nm2 per molecule. For NaDC, ΔCp demic decreases at 343 K to ∼250 J·mol-1·K-1, whereas ΔCp demic for NaC remains essentially unchanged. The calorimetric titration curves were simulated using a mass action model including counterion condensation for the aggregation process. The simulation of the titration curves yielded values for the aggregation number n. In the concentration region of the cmc, n is approximately 4−6 for NaC in water or 0.1 M NaCl and independent of temperature. For NaDC in water values of n of 7 and 12 were obtained at low temperature (284 K) in water and 0.1 M NaCl, respectively. For NaDC in water and 0.1 M NaCl, the aggregation number n decreases to 5 and 7, respectively, at 328 K.
Aggregation of ␣-synuclein is a key event in several neurodegenerative diseases, including Parkinson disease. Recent findings suggest that oligomers represent the principal toxic aggregate species. Using confocal single-molecule fluorescence techniques, such as scanning for intensely fluorescent targets (SIFT) and atomic force microscopy, we monitored ␣-synuclein oligomer formation at the single particle level. Organic solvents were used to trigger aggregation, which resulted in small oligomers ("intermediate I"). Under these conditions, Fe 3؉ at low micromolar concentrations dramatically increased aggregation and induced formation of larger oligomers ("intermediate II"). Both oligomer species were on-pathway to amyloid fibrils and could seed amyloid formation. Notably, only Fe 3؉ -induced oligomers were SDS-resistant and could form ion-permeable pores in a planar lipid bilayer, which were inhibited by the oligomerspecific A11 antibody. Moreover, baicalein and N-benzylidenebenzohydrazide derivatives inhibited oligomer formation. Baicalein also inhibited ␣-synuclein-dependent toxicity in neuronal cells. Our results may provide a potential disease mechanism regarding the role of ferric iron and of toxic oligomer species in Parkinson diseases. Moreover, scanning for intensely fluorescent targets allows high throughput screening for aggregation inhibitors and may provide new approaches for drug development and therapy.
Within the European Immunogenicity Platform (EIP) (http://www.e-i-p.eu), the Protein Characterization Subcommittee (EIP-PCS) has been established to discuss and exchange experience of protein characterization in relation to unwanted immunogenicity. In this commentary, we, as representatives of EIP-PCS, review the current state of methods for analysis of protein aggregates. Moreover, we elaborate on why these methods should be used during product development and make recommendations to the biotech community with regard to strategies for their application during the development of protein therapeutics.
SummaryBacteria have evolved elaborate communication strategies to co-ordinate their group activities, a process termed quorum sensing (QS). Pseudomonas aeruginosa is an opportunistic pathogen that utilizes QS for diverse activities, including disease pathogenesis. P. aeruginosa has evolved a novel communication system in which the signal molecule 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas Quinolone Signal, PQS) is trafficked between cells via membrane vesicles (MVs). Not only is PQS packaged into MVs, it is required for MV formation. Although MVs are involved in important biological processes aside from signalling, the molecular mechanism of MV formation is unknown. To provide insight into the molecular mechanism of MV formation, we examined the interaction of PQS with bacterial lipids. Here, we show that PQS interacts strongly with the acyl chains and 4Ј-phosphate of bacterial lipopolysaccharide (LPS). Using PQS derivatives, we demonstrate that the alkyl side-chain and third position hydroxyl of PQS are critical for these interactions. Finally, we show that PQS stimulated purified LPS to form liposome-like structures. These studies provide molecular insight into P. aeruginosa MV formation and demonstrate that quorum signals serve important non-signalling functions.
Bacterial endotoxins (lipopolysaccharides (LPS)) are strong elicitors of the human immune system by interacting with serum and membrane proteins such as lipopolysaccharide-binding protein (LBP) and CD14 with high specificity. At LPS concentrations as low as 0.3 ng/ml, such interactions may lead to severe pathophysiological effects, including sepsis and septic shock. One approach to inhibit an uncontrolled inflammatory reaction is the use of appropriate polycationic and amphiphilic antimicrobial peptides, here called synthetic anti-LPS peptides (SALPs). We designed various SALP structures and investigated their ability to inhibit LPS-induced cytokine secretion in vitro, their protective effect in a mouse model of sepsis, and their cytotoxicity in physiological human cells. Using a variety of biophysical techniques, we investigated selected SALPs with considerable differences in their biological responses to characterize and understand the mechanism of LPS inactivation by SALPs. Our investigations show that neutralization of LPS by peptides is associated with a fluidization of the LPS acyl chains, a strong exothermic Coulomb interaction between the two compounds, and a drastic change of the LPS aggregate type from cubic into multilamellar, with an increase in the aggregate sizes, inhibiting the binding of LBP and other mammalian proteins to the endotoxin. At the same time, peptide binding to phospholipids of human origin (e.g., phosphatidylcholine) does not cause essential structural changes, such as changes in membrane fluidity and bilayer structure. The absence of cytotoxicity is explained by the high specificity of the interaction of the peptides with LPS.
Isothermal titration calorimetry (ITC) was used to investigate the interactions of bile salts with phosphatidylcholine vesicles. We determined the partition coefficients of the detergents sodium cholate (NaC) and sodium deoxycholate (NaDC) and the respective transfer enthalpies between pure water or 0.1 M aqueous salt solution and bilayers, consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) and of 1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine (DMPC). Additionally, the vesicle-to-micelle transition was investigated for NaC/DPPC and NaDC/DPPC systems in water and 0.1 M NaCl. ITC was employed to determine the phase boundaries for the saturation and the complete solubilization of the vesicles by the bile salts enclosing the coexistence region of mixed vesicles and micelles. To study the influence of the alkyl chain length of the phospholipids on the phase behavior we also studied the NaDC/ DMPC system. Saturated phosphatidylcholines are more easily transformed into micelles than those with unsaturated chains. In the region of low lipid concentrations we observed a departure from linearity of the phase boundaries, which was explained by the influence of the energy of end-caps as proposed by Roth et al. (Langmuir 2000(Langmuir , 16, 2052. The deviation was larger for systems in pure water compared to those in 0.1 M NaCl. The saturation concentrations of bilayers of DPPC and DMPC were much lower than those for unsaturated analogues. The saturation concentration increased with increasing salt content, and the coexistence range became wider. The ITC solubilization curves could be analyzed by applying the known values for partition coefficients and transfer enthalpies for the detergents to the different types of aggregates.
IR spectroscopic studies are reported for the phytosphingosine class of ceramides and are compared with two analogous sphingosine ceramides. The phytosphingosine class of molecules, not previously widely investigated by physical techniques, constitutes ∼30% of the total ceramide content of the stratum corneum, the barrier to permeability in skin. The current measurements utilize temperature-controlled horizontal attenuated total reflectance spectroscopy of hydrated films to study H f D exchange in the polar regions of the molecules as well as chain conformational order and packing properties. Analysis of the methylene stretching and scissoring vibrations reveals that the chains of the two phytosphingosine derivatives (ceramides 3 and 7) are much more poorly packed at room temperature than their sphingosine counterparts (ceramides 2 and 5 respectively), despite having order f disorder transitions some 15-20 degrees higher. This unanticipated relative stability of the phytosphingosines is traced to enhanced headgroup H-bonding interactions manifest by lower Amide I and higher Amide II frequencies. Water penetration into the polar regions is monitored by the temperature dependence of the Amide II and O-H/N-H stretching intensities as a function of HfD exchange. Neither ceramide 2 nor 3 exchanges N-H or O-H protons until relatively high temperatures (>65 °C). However, addition of an R-hydroxy group on the fatty acid chain in ceramides 5 or 7 results in exchange events observed at temperatures much closer to physiological. These measurements reveal that the relative contributions of chain packing and H-bonding under physiological conditions differ markedly for the phytosphingosines compared to the sphingosines. The former are characterized by hexagonal chain packing with relatively strong H-bonding; the latter by orthorhombic chain packing and weaker H-bonding. The implications of these molecular structure data for lipid organization in the stratum corneum are briefly discussed.
The miscibilities of phosphatidic acids (PAs) and phosphatidylcholines (PCs) with different chain lengths (n = 14, 16) at pH 4, pH 7, and pH 12 were examined by differential scanning calorimetry. Simulation of heat capacity curves was performed using a new approach that incorporates changes of cooperativity of the transition in addition to nonideal mixing in the gel and the liquid-crystalline phase as a function of composition. From the simulations of the heat capacity curves, first estimates for the nonideality parameters for nonideal mixing as a function of composition were obtained, and phase diagrams were constructed using temperatures for onset and end of melting, which were corrected for the broadening effect caused by a decrease in cooperativity. In all cases the composition dependence of the nonideality parameters indicated nonsymmetrical mixing behavior. The phase diagrams were therefore further refined by simulations of the coexistence curves using a four-parameter approximation to account for nonideal and nonsymmetrical mixing in the gel and the liquid-crystalline phase. The mixing behavior was studied at three different pH values to investigate how changes in headgroup charge of the PA influences the miscibility. The experiments showed that at pH 7, where the PA component is negatively charged, the nonideality parameters are in most cases negative, indicating that electrostatic effects favor a mixing of the two components. Partial protonation of the PA component at pH 4 leads to strong changes in miscibility; the nonideality parameters for the liquid-crystalline phase are now in most cases positive, indicating clustering of like molecules. The phase diagram for 1,2-dimyristoyl-sn-glycero-3-phosphatidic acid:1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine mixtures at pH 4 indicates that a fluid-fluid immiscibility is likely. The results show that a decrease in ionization of PAs can induce large changes in mixing behavior. This occurs because of a reduction in electrostatic repulsion between PA headgroups and a concomitant increase in attractive hydrogen bonding interactions.
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