A critical concentration of N-terminal pyroglutamylated amyloid beta drives the misfolding of Ab1-42 into more toxic aggregates.

A wide consensus based on robust experimental evidence indicates pyroglutamylated amyloid-ß isoform (AßpE3-42) as one of the most neurotoxic peptides involved in the onset of Alzheimer's disease. Furthermore, AßpE3-42 co-oligomerized with excess of Aß1-42, produces oligomers and aggregates that are structurally distinct and far more cytotoxic than those made from Aß1-42 alone. Here, we investigate quantitatively the influence of AßpE3-42 on biophysical properties and biological activity of Aß1-42. We tested different ratios of AßpE3-42/Aß1-42 mixtures finding a correlation between the biological activity and the structural conformation and morphology of the analyzed mixtures. We find that a mixture containing 5% AßpE3-42, induces the highest disruption of intracellular calcium homeostasis and the highest neuronal toxicity. These data correlate to an high content of relaxed antiparallel ß-sheet structure and the coexistence of a population of big spheroidal aggregates together with short fibrils. Our experiments provide also evidence that AßpE3-42 causes template-induced misfolding of Aß1-42 at ratios below 33%. This means that there exists a critical concentration required to have seeding on Aß1-42 aggregation, above this threshold, the seed effect is not possible anymore and AßpE3-42 controls the total aggregation kinetics.

Electrostatic theory of the assembly of PAMAM dendrimers and DNA.

The electrostatic interactions mediated by counterions between a cationic PAMAM dendrimer, modelized as a sphere of radius and cationic surface charge highly increasing with generation, and a DNA, modelized as an anionic elastic line, are analytically calculated in the framework of condensation theory. Under these interactions the DNA is wrapped around the sphere. For excess phosphates relative to dendrimer primary amines, the free energy of the DNA-dendrimer complex displays an absolute minimum when the complex is weakly negatively overcharged. This overcharging opposes gene delivery. For a highly positive dendrimer and a DNA fixed by experimental conditions to a number of phosphates less than the number of dendrimer primary amines, excess amine charges, the dendrimer may at the same time bind stably DNA and interact with negative cell membranes to activate cell transfection in fair agreement with molecular simulations and experiments.

Lamellar cationic lipid-DNA complexes from lipids with a strong preference for planar geometry: A Minimal Electrostatic Model.

We formulate and analyze a minimal model, based on condensation theory, of the lamellar cationic lipid (CL)-DNA complex of alternately charged lipid bilayers and DNA monolayers in a salt solution. Each lipid bilayer, composed by a random mixture of cationic and neutral lipids, is assumed to be a rigid uniformly charged plane. Each DNA monolayer, located between two lipid bilayers, is formed by the same number of parallel DNAs with a uniform separation distance. For the electrostatic calculation, the model lipoplex is collapsed to a single plane with charge density equal to the net lipid and DNA charge. The free energy difference between the lamellar lipoplex and a reference state of the same number of free lipid bilayers and free DNAs, is calculated as a function of the fraction of CLs, of the ratio of the number of CL charges to the number of negative charges of the DNA phosphates, and of the total number of planes. At the isoelectric point the free energy difference is minimal. The complex formation, already favoured by the decrease of the electrostatic charging free energy, is driven further by the free energy gain due to the release of counterions from the DNAs and from the lipid bilayers, if strongly charged. This minimal model compares well with experiment for lipids having a strong preference for planar geometry and with major features of more detailed models of the lipoplex.

Differential toxicity, conformation and morphology of typical initial aggregation states of Aß1-42 and Aßpy3-42 beta-amyloids.

Among the different species of water-soluble ß-peptides (Aß1-42, Aß1-40 and N-terminal truncated Aß-peptides), Aßpy3-42 is thought to play a relevant role in Alzheimer's pathogenesis due to its abundance, resistance to proteolysis, fast aggregation kinetics, dynamic structure and high neurotoxicity. To evaluate the specific structural characteristics and neurotoxicity of Aßpy3-42, we separated different aggregation states of Aß1-42 and Aßpy3-42 using fast protein liquid chromatography, isolating in both cases three peaks that corresponded to sa (small), ma (medium) and la (large) aggregates. Conformational analysis, by circular dichroism showed a prevailing random coil conformation for sa and ma, and typical ß-sheet conformation for la. AFM and TEM show differential structural features between the three aggregates of a given ß-peptide and among the aggregate of the two ß-peptides. The potential toxic effects of the different aggregates were evaluated using human neuroblastoma SH-SY5Y cells in the MTT reduction, in the xCELLigence System, and in the Annexin V binding experiments. In the case of Aß1-42 the most toxic aggregate is la, while in the case of Aßpy3-42 both sa and la are equally toxic. Aß aggregates were found to be internalized in the cells, as estimated by confocal immunofluorescence microscopy, with a higher effect observed for Aßpy3-42, showing a good correlation with the toxic effects. Together these experiments allowed the discrimination of the intermediate states more responsible of oligomer toxicity, providing new insights on the correlation between the aggregation process and the toxicity and confirming the peculiar role in the pathogenesis of Alzheimer disease of Aßpy3-42 peptide.

Clusters in strong polyelectrolyte solutions in the condensation theory approach.

The interaction free energy of parallel clusters of like-charged rod polyelectrolytes in solution is calculated in the framework of the extended condensation theory. For sufficiently high linear charge density of the polyelectrolyte, clustering takes place. The greater is the number of polyelectrolytes participating to the cluster, the smaller is the equilibrium interpolyelectrolyte distance, and the deeper is the corresponding free energy minimum. It is a counterintuitive organization due to the increasing of the counterion condensed charge and condensation volume, taking place as the polyelectyrolytes approach each other.

Wrapped-around models for the lac operon complex.

The protein-DNA complex, involved in the lac operon of enteric bacteria, is paradigmatic in understanding the extent of DNA bending and plasticity due to interactions with protein assemblies acting as DNA regulators. For the lac operon, two classes of structures have been proposed: 1), with the protein tetramer lying away from the DNA loop (wrapped-away model); and 2), with the protein tetramer lying inside the DNA loop (wrapped-around model). A recently developed electrostatic analytical model shows that the size and net charge of the Lac protein tetramer allow the bending of DNA, which is consistent with another wrapped-around model from the literature. Coarse-grained models, designed based on this observation, are extensively investigated and show three kinds of wrapped-around arrangements of DNA and a lower propensity for wrapped-away configurations. Molecular dynamics simulations of an all-atom model, built on the basis of the most tightly collapsed coarse-grained model, show that most of the DNA double-helical architecture is maintained in the region between O3 and O1 DNA operators, that the DNA distortion is concentrated in the chain beyond the O1 operator, and that the protein tetramer can adapt the N-terminal domains to the DNA tension.

N-terminal truncated pyroglutamyl beta amyloid peptide Abetapy3-42 shows a faster aggregation kinetics than the full-length Abeta1-42.

We tested directly the differences in the aggregation kinetics of three important beta amyloid peptides, the full-length Abeta1-42, and the two N-terminal truncated and pyroglutamil modified Abetapy3-42 and Abetapy11-42 found in different relative concentrations in the brains in normal aging and in Alzheimer disease. By following the circular dichroism signal and the ThT fluorescence of the solution in phosphate buffer, we found substantially faster aggregation kinetics for Abetapy3-42. This behavior is due to the particular sequence of this peptide, which is also responsible for the specific oligomeric aggregation states, found by TEM, during the fibrillization process, which are very different from those of Abeta1-42, more prone to fibril formation. In addition, Abetapy3-42 is found here to have an inhibitory effect on Abeta1-42 fibrillogenesis, coherently with its known greater infective power. This is an indication of the important role of this peptide in the aggregation process of beta-peptides in Alzheimer disease.

The supramolecular association of polyelectrolytes to complementary charged surfactants and protein assemblies.

Cohesion matters! The correlation between the conformational rigidity of the polyelectrolyte and the size and stability of the globular assembly is discussed in this review article. Some examples of models for the association of polyelectrolytes to globular assemblies are shown here.Supramolecular complexes of strong polyelectrolytes and oppositely charged ionic micelles or protein assemblies derive their main stabilization from electrostatic interactions that include the counterion condensation/release mechanism and from hydrophobic interactions distributed along apolar sections of the components. The predicted and the experimental behavior of selected complexes differing in the flexibility of the polyelectrolyte and in the cohesion of the complementary assembly is reviewed and analyzed. Depending upon the rigidity of the polyelectrolyte, globular surfactant clusters persist in the final structure or are transformed into elongated assemblies. On the other hand, even the rather rigid DNA molecule is forced to bend by strongly associated cationic protein complexes such as the histone octamers. A general framework should allow the prediction of these structures in terms of the interplay between the persistence length of the polyelectrolyte and the association constant of the protein or the surfactant assembly.

Attraction between like-charged polyelectrolytes in the extended condensation theory.

The interaction free energy of like-charged polyelectrolytes in solution is calculated in the framework of the extended counterion condensation theory, recently given by Schurr and Fujimoto, Biophys. Chem. 2002, 101-102, 425-445. For sufficiently high linear charge density, the electrostatic free energy of two parallel identical rigid polyelectrolytes as a function of the distance between them shows a minimum at distances in the range of nanometers, increasing with the Debye screening length. This effect is due to the increasing of the counterion condensed charge and condensation volume as the two polyelectyrolytes approach.

Intracellular accumulation of a mild-denatured monomer of the human PrP fragment 90-231, as possible mechanism of its neurotoxic effects.

Because of high tendency of the prion protein (PrP) to aggregate, the exact PrP isoform responsible for prion diseases as well as the pathological mechanism that it activates remains still controversial. In this study, we show that a pre-fibrillar, monomeric or small oligomeric conformation of the human PrP fragment 90-231 (hPrP90-231), rather than soluble or fibrillar large aggregates, represents the neurotoxic species. In particular, we demonstrate that monomeric mild-denatured hPrP90-231 (incubated for 1 h at 53 degrees C) induces SH-SY5Y neuroblastoma cell death, while, when structured in large aggregates, it is ineffective. Using spectroscopic and cellular techniques we demonstrate that this toxic conformer is characterized by a high exposure of hydrophobic regions that favors the intracellular accumulation of the protein. Inside the cells hPrP90-231 is mainly compartmentalized into the lysosomes where it may trigger pro-apoptotic 'cell death' signals. The PrP toxic conformation, which we have obtained inducing a controlled in vitro conformational change of the protein, might mimic mild-unfolding events occurring in vivo, in the presence of specific mutations, oxidative reactions or proteolysis. Thus, in light of this model, we propose that novel therapeutic strategies, designed to inhibit the interaction of the toxic PrP with the plasmamembrane, could be beneficial to prevent the formation of intracellular neurotoxic aggregates and ultimately the neuronal death.

Generalized electrostatic model of the wrapping of DNA around oppositely charged proteins.

Histonelike proteins in prokaryotes and histone octamers in eukaryotes carry large positive charges, which are responsible of strong electrostatic interactions with DNA. As a result, DNA wraps around proteins and genetic information is condensed. We describe a generalized model of these electrostatic interactions mediated by salt that explains the wrapping of DNA around the nucleosome octamer, around remodeling factors in eukaryotes and around histonelike proteins in prokaryotes. It comes out that small changes in protein dimension and charge produce large effects in the supramolecular DNA-protein architecture.

Modeling H3 histone N-terminal tail and linker DNA interactions.

Molecular dynamics computer simulations were performed for the 25-residue N-terminal tail of the H3 histone protein in the proximity of a DNA segment of 10 base pairs (bp), representing a model for the linker DNA in chromatin. Several least biased configurations were used as initial configurations. The secondary structure content of the protein was increased by the presence of DNA close to it, but the locations of the secondary motifs were different for different initial orientations of the DNA grooves with respect to the protein. As a common feature to all simulations, the electrostatic attraction between negatively charged DNA and positively charged protein was screened by the water solvent and counterbalanced by the intrinsic compaction of the protein due to hydrophobic effects. The protein secondary structure limited the covering of DNA by the protein to 4-5 bp. The degree of compaction and charge density of the bound protein suggests a possible role of H3 tail in a nonspecific bending and plasticity of the linker DNA when the protein is located in the crowded dense chromatin.

Electrostatic interactions with histone tails may bend linker DNA in chromatin.

Is linker DNA bent in the 30-nm chromatin fiber at physiological conditions? We show here that electrostatic interactions between linker DNA and histone tails including salt condensation and release may bend linker DNA, thus affecting the higher order organization of chromatin.

Hyaluronan chain conformation and dynamics.

An overview of the present state of research in the field of hyaluronan chain conformational aspects is presented. The relationship between structure and dynamics are illustrated for a series of hyaluronan oligomers. Conformational characteristics of hyaluronan chains are discussed, together with the dynamic chain patterns, evaluated by using a theoretical approach to diffusive polymer dynamics. The dependence of correlation times and NMR relaxation parameters from the chain dimension are investigated. Topological features and dimensional properties are related to the structural determinants by using classical computational methods of molecular mechanics and Monte Carlo simulation.

Designing generalized statistical ensembles for numerical simulations of biopolymers.

Conformational properties of polymers, such as average dihedral angles or molecular alpha-helicity, display a rather weak dependence on the detailed arrangement of the elementary constituents (atoms). We propose a computer simulation method to explore the polymer phase space using a variant of the standard multicanonical method, in which the density of states associated to suitably chosen configurational variables is considered in place of the standard energy density of states. This configurational density of states is used in the Metropolis acceptance/rejection test when configurations are generated with the help of a hybrid Monte Carlo algorithm. The resulting configurational probability distribution is then modulated by exponential factors derived from the general principle of the maximal constrained entropy by requiring that certain average configurational quantities take preassigned (possibly temperature dependent) values. Thermal averages of other configurational quantities can be computed by using the probability distributions obtained in this way. Moments of the energy distribution require an extra canonical sampling of the system phase space at the desired temperature, in order to locally thermalize the configurational degrees of freedom. As an application of these ideas we present the study of the structural properties of two simple models: a bead-and-spring model of polyethylene with independent hindered torsions and an all-atom model of alanine and glycine oligomers with 12 amino acids in vacuum.

Modeling the backbone dynamics of reduced and oxidized solvated rat microsomal cytochrome b5.

In this article, a description of the statistics and dynamics of cytochrome b(5) in both reduced and oxidized forms is given. Results of molecular dynamics computer simulations in the explicit solvent have been combined with mode-coupling diffusion models including and neglecting the molecule-solvent correlations. R(1) and R(1 rho) nuclear magnetic relaxation parameters of (15)N in the protein backbone have been calculated and compared with experiments. Slight changes in charge density in the heme upon oxidation produces a cascade of changes in charge distributions from heme propionates up to charged residues approximately 1.5 nm from Fe. These changes in charge distributions modify the molecular surface and the water shell surrounding the protein. The statistical changes upon oxidation can be included in diffusive models that physically explain the upper and lower limits of R(1 rho) relaxation parameters at high off-resonance fields.