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Diabetic neuropathy (DN) is a long-term complication of diabetes mellitus, affecting different periphery nerve systems including sensory and motor neurons. Hyperglycemia is the major cause of DN with symptoms such as weakness of balance or coordination, insensitivity to sensation, weakness of the muscles as well as numbness and pain in limbs Analgesic drug such as opioids can be effective to relief neuropathy pain but there is no effective treatment. Adiponectin is an anti-diabetic adipokine, which possesses insulin-sensitizing and neuroprotective effects. In this project, we aim to identify an agent which is dual acting to opioid and adiponectin receptors. Within a virtual screening repositioning campaign, a large collection of compounds with different structures comprehensive of adipoRon-like piperidine derivatives was screened by docking. Recently developed opioid receptor benzomorphanic agonists finally emerged as good ligands to adiponectin receptors showing some 2D and 3D structural similarities with AdipoRon. Particularly, we have identified (+)-MML1017, which has high affinity to the same binding domain of AdipoR1 and AdipoR2 as AdipoRon. Our western blot results indicate (+)-MML1017 activates AMPK phosphorylation through both adipoR1 and adipoR2 in neuronal cell line. Moreover, pretreatment of (+)-MML1017 can improve the cell viability with motor neurons under hyperglycermic conditions. The (+)-MML1017 also activates μ-opioid receptor cells in a concentration-dependent manner. Our study identified a novel compound having dual activity on opioid receptors and adiponectin receptors that may have analgesic effects and neuroprotective effects to treat diabetic neuropathy.

It is well known that flavanones have beneficial health effects. Eriodictyol-7-rutinoside, also called eriocitrin, is a flavanone with many positive effects. Like many of these compounds, low water solubility, stability and shelf life are major issues. Many studies have focused on improving these aspects. Towards this goal, β-cyclodextrin and its derivatives are interesting candidates. These compounds are characterized by a hydrophobic central cavity that usually enhances ligand solubility in aqueous solutions, affecting the chemical characteristics of the encapsulated ligand. Recently, eriocitrin/hydroxypropyl-β-cyclodextrin complex with advanced properties has been reported. Herein, computational approaches were combined with NMR spectroscopy to characterize the eriocitrin/β-cyclodextrin complex and compare it with that of eriocitrin/hydroxypropyl-β-cyclodextrin.

Background: Central and peripheral analgesia without adverse effects relies on the identification of μ-opioid agonists that are able to activate ‘basal’ antinociceptive pathways. Recently developed μ-selective benzomorphan agonists that are not antagonized by naloxone do not activate G-proteins and β-arrestins. Which pathways do μ receptors activate? How can each of them be selectively activated? What role is played by allosteric binding sites? Methodology & results: Molecular modeling studies characterize the amino acid residues involved in the interaction with various classes of endogenous and exogenous ligands and with agonists and antagonists. Conclusions: Critical binding differences between various classes of agonists with different pharmacological profiles have been identified. MML series binding poses may be relevant in the search for an antinociception agent without side effects.

Functional antitumor vaccine constructs are the basis for active tumor immunotherapy, which is useful in the treatment of many types of cancers. MUC1 is one key glycoprotein for targeting and designing new strategies for multicomponent vaccines. Two self-adjuvant tetravalent vaccine candidates were prepared by clustering four or eight PDTRP MUC1 core epitope sequences on calixarene scaffolds. In this work, the different activities of two molecules with calix[4]arene and calix[8]arene skeleton are rationalized. Quantum mechanics, docking, and molecular dynamics structural optimization were first carried out followed by metadynamics to calculate the energy profiles. Further insights were obtained by complementarity studies of molecular fields. The molecular modeling results are in strong agreement with the experimental in vivo immunogenicity data. In conclusion, the overall data shows that, in the designing of anticancer vaccines, scaffold flexibility has a pivotal role in obtaining a suitable electrostatic, hydrophobic, and steric complementarity with the biological target.

G protein-coupled receptors (GPCRs) are a super-family of membrane proteins that attract great pharmaceutical interest due to their involvement in almost every physiological activity, including extracellular stimuli, neurotransmission, and hormone regulation. Currently, structural information on many GPCRs is mainly obtained by the techniques of computer modelling in general and by homology modelling in particular. Based on a quantitative analysis of eighteen antagonist-bound, resolved structures of rhodopsin family "A" receptors - also used as templates to build 153 homology models - it was concluded that a higher sequence identity between two receptors does not guarantee a lower RMSD between their structures, especially when their pair-wise sequence identity (within trans-membrane domain and/or in binding pocket) lies between 25 % and 40 %. This study suggests that we should consider all template receptors having a sequence identity ≤50 % with the query receptor. In fact, most of the GPCRs, compared to the currently available resolved structures of GPCRs, fall within this range and lack a correlation between structure and sequence. When testing suitability for structure-based drug design, it was found that choosing as a template the most similar resolved protein, based on sequence resemblance only, led to unsound results in many cases. Molecular docking analyses were carried out, and enrichment factors as well as attrition rates were utilized as criteria for assessing suitability for structure-based drug design.

Endogenous peptides as part of physiological processes are targets of interest when it comes to finding desirable therapeutics which are able to modulate molecular interactions. The major limits presented by peptides when they are used as drugs have motivated the research of the synthesis of peptidomimetics obtained through chemical modification and the use of in silico approaches. Here recent works on the discovery of peptidomimetics by computational methods are reported. Together with molecular dynamic simulations, the use of pharmacophore research simulations helps to gain insight into and understand the molecular determinants underlying the physiological processes.

In this paper, the techniques of modelling, docking and molecular dynamics were used to study eight single amino acid mutations of the enzyme PhnI to optimise its enzymatic degradation capability. The eight mutants were first equilibrated to avoid deformations of the secondary and tertiary structure and to minimise alterations in the functionality of the chimera enzymes that were obtained. For this purpose, we monitored the potential energy of the systems and the fluctuations of the backbone of the enzymes. The structures of mutant enzymes, at equilibrium, were subjected to docking calculations with selected PAHs. The results indicated a significant increase in the PAH-enzyme interaction with respect to the wild-type protein. The considerable computing resources offered by the GRID computing system made it possible to perform calculations on the entire enzyme system, consisting of six protein subunits, as highlighted in the recent literature.

Aggregation of Islet amyloid polypeptide (IAPP) is believed to play a critical role in the pathogenesis of Type II Diabetes Mellitus. In an attempt to gain details on the early events of this process, here we performed MD simulations of the spontaneous assembly of three replicas of human IAPP. Systems containing the Cys2-Cys7 disulfide bridge exhibited a greater stability and a decreased tendency to evolve into β-sheet rich structures if compared to the disulfide-depleted variants. Conversely, the stability of assemblies constituted by the rat isoforms was shown to be independent from the presence of the disulfide bridge.

Agricultural practices are usually supported by several chemical substances, such as herbicides. Linuron and chlorbromuron are phenylurea herbicides largely used to protect crops from weeds, blocking photosynthesis by inhibition of the photosystem II complex. The former, also commercially known as lorox or afalon, is selectively used to protect bean and French bean plants, fennels, and celeriacs; the second, commercially known as maloran, is selectively used for carrots, peas, potatoes, soy sprouts, and sunflowers. Considering the widespread use of herbicides and, more generally, pesticides, it is important to clarify their involvement on human health, one of them concerning the possible direct or indirect effect on the genome of exposed populations. Here, we show that these herbicides are endowed by mutagenic properties, as demonstrated by an increased number of chromosomal aberrations (CAs) in two exposed Chinese hamster cell lines derived from ovary and epithelial liver, respectively. This was also confirmed by sister chromatid exchange (SCE) and micronucleus (MN) assays. Our present and previously obtained data clearly indicate that phenylurea herbicides must be used with great caution, especially for agricultural workers who use large amounts of herbicides during their work, and particular attention should be given to residues of these herbicides and their involvement in environmental pollution.

Experimental and computational studies suggest that few general principles govern protein/protein interactions and aggregation. The knowledge of these rules may be exploited to design peptides that are able to interfere with the self-assembly and aggregation of proteins. This work is aimed to verify the validity of this hypothesis by investigating the interaction of cytochrome c with Phe and Gly amino acids, Ala-His (carnosine), and two water-soluble dipeptides Phe-Gly and Gly-Phe. The combined use of 1 H NMR, MD, and DSC has shown that: (i) at neutral pH, only Phe-Gly is able to prevent the thermally induced aggregation of cytochrome c; (ii) Phe-Gly interacts with Gly45 and Phe46 residues of the protein, either when the protein is in the folded or in the unfolded state; and (iii) the interaction of Phe-Gly with cytochrome c is sequence-dependent. These results support the hypothesis that the basic principles that describe protein aggregation can be used for the design of peptides with antiaggregating properties.  2004 Published by Elsevier Inc.

Modeling G-Protein Coupled Receptors (GPCRs) is an emergent field of research, since utility of high-quality models in receptor structure-based strategies might facilitate the discovery of interesting drug candidates. The findings from a quantitative analysis of eighteen resolved structures of rhodopsin family "A" receptors crystallized with antagonists and 153 pairs of structures are described. A strategy termed endeca-amino acids fragmentation was used to analyze the structures models aiming to detect the relationship between sequence identity and Root Mean Square Deviation (RMSD) at each trans-membrane-domain. Moreover, we have applied the leave-one-out strategy to study the shiftiness likelihood of the helices. The type of correlation between sequence identity and RMSD was studied using the aforementioned set receptors as representatives of membrane proteins and 98 serine proteases with 4753 pairs of structures as representatives of globular proteins. Data analysis using fragmentation strategy revealed that there is some extent of correlation between sequence identity and global RMSD of 11AA width windows. However, spatial conservation is not always close to the endoplasmic side as was reported before. A comparative study with globular proteins shows that GPCRs have higher standard deviation and higher slope in the graph with correlation between sequence identity and RMSD. The extracted information disclosed in this paper could be incorporated in the modeling protocols while using technique for model optimization and refinement.

Amylin is a 37-residue peptide hormone produced by the islet beta-cells of pancreas and the formation of amylin aggregates is strongly associated with beta-cell degeneration in type 2 diabetes, as demonstrated by more than 95% of patients exhibiting amylin amyloid upon autopsy. It is widely recognized that metal ions such as copper(II) have been implicated in the aggregation process of amyloidogenic peptides such as Ab and alpha-synuclein and there is evidence that amylin self-assembly is also largely affected by copper(II). For this reason, in this work, the role of copper(II) in the aggregation of amylin has been investigated by several different experimental approaches. Mass spectrometric investigations show that copper(II) induces significant changes in the amylin structure, which decrease the protein fibrillogenesis as observed by ThT measurements. Accordingly, solid-state NMR experiments together with computational analysis carried out on a model amylin fragment confirmed the non-fibrillogenic nature of the copper(II) induced aggregated structure. Finally, the presence of copper(II) is also shown to have a major influence on amylin proneness to be degraded by proteases and cytotoxicity studies on different cell cultures are reported.

Imidazoline ligands in I2-type binding sites in the brain alter monoamine turnover and release. One example of an I2 binding site characterized by binding studies, kinetics, and crystal structure has been described in monoamine oxidase B (MAO B). MAO A also binds imidazolines but has a different active site structure. Docking and molecular dynamics were used to explore how 2-(2-benzofuranyl)-2-imidazoline hydrochloride (2-BFI) binds to MAO A and to explain why tranylcypromine increases tight binding to MAO B. The energy for 2-BFI binding to MAO A was comparable to that for tranylcypromine-modified MAO B, but the location of 2-BFI in the MAO A could be anywhere in the monopartite substrate cavity. Binding to the tranylcypromine-modified MAO B was with high affinity and in the entrance cavity as in the crystal structure, but the energies of interaction with the native MAO B were less favorable. Molecular dynamics revealed that the entrance cavity of MAO B after tranylcypromine modification is both smaller and less flexible. This change in the presence of tranylcypromine may be responsible for the greater affinity of tranylcypromine-modified MAO B for imidazoline ligands.

Polycyclic aromatic hydrocarbons are a family of ubiquitous pollutants whose environmental behavior has been widely studied. Different bacterial species are able to decompose hydrocarbons by using them as a food source. One of the best-studied enzymes is naphthalene 1,2-dioxygenase (NDO). A practical way to optimize the degradation process is by mutating the protein involved, increasing both the degradation capacity of the enzyme and its ability to work under extreme environmental conditions of high temperature and low pH. Herein, we describe the study of NDO using molecular dynamics and docking calculations to discover new mutants with high degrading capabilities. We modeled eleven new mutants of NDO. The results indicate that increasing the size of the active site cavity in the mutants allowed for the insertion of high molecular weight PAHs. Additionally, the physicochemical properties of the NDO active sites make the sites well suited to interactions with PAHs, so most amino-acid modifications should not result in significantly altered behavior of NDO.

The human histamine H-4 receptor (hH(4)R), a member of the G-protein coupled receptors (GPCR) family, is an increasingly attractive drug target. It plays a key role in many cell pathways and many hH(4)R ligands are studied for the treatment of several inflammatory, allergic and autoimmune disorders, as well as for analgesic activity. Due to the challenging difficulties in the experimental elucidation of hH(4)R structure, virtual screening campaigns are normally run on homology based models. However, a wealth of information about the chemical properties of GPCR ligands has also accumulated over the last few years and an appropriate combination of these ligand-based knowledge with structure-based molecular modeling studies emerges as a promising strategy for computer-assisted drug design. Here, two chemoinformatics techniques, the Intelligent Learning Engine (ILE) and Iterative Stochastic Elimination (ISE) approach, were used to index chemicals for their hH(4)R bioactivity. An application of the prediction model on external test set composed of more than 160 hH(4)R antagonists picked from the chEMBL database gave enrichment factor of 16.4. A virtual high throughput screening on ZINC database was carried out, picking similar to 4000 chemicals highly indexed as H4R antagonists' candidates. Next, a series of 3D models of hH(4)R were generated by molecular modeling and molecular dynamics simulations performed in fully atomistic lipid membranes. The efficacy of the hH(4)R 3D models in discrimination between actives and non-actives were checked and the 3D model with the best performance was chosen for further docking studies performed on the focused library. The output of these docking studies was a consensus library of 11 highly active scored drug candidates. Our findings suggest that a sequential combination of ligand-based chemoinformatics approaches with structure-based ones has the potential to improve the success rate in discovering new biologically active GPCR drugs and increase the enrichment factors in a synergistic manner.

In recent years, the number of studies in the field of bioremediation has been growing steadily. Although a large number of studies provide information that is highly detailed and offer great amounts of knowledge on a given subject, the downside is that the hunt for more information requires the combined efforts of researchers from many areas, which are becoming increasingly difficult to attain. In this review, we present an overview of recent work investigating enzyme degradation of polycyclic aromatic hydrocarbons. In the first part, this review examines several of the new enzymes able to degrade pollutants, with special attention being given to those with a well-resolved structure. The second part explores some of the most recent work in which computational approaches, such as molecular dynamics, docking, density functional theory and database retrieval, have been employed to study enzymes with specific bioremediation activities.

The hydrosoluble resveratrol derivative 3-O-phosphorylresveratrol was shown to be more cytotoxic against DU 145 prostate cancer cells than its analog 4'-O-phosphorylresveratrol. In an attempt to unveil the molecular determinants that lye at the root of their different biological effects, here we investigate the interactions of the two resveratrol derivatives with DMPC model membranes by using DSC, membrane permeation/poration assays and molecular dynamics. The results show that the 3-O-derivative interacts with DMPC membranes and diffuses across them. The 4'-O-derivative lies preferentially onto the surface of membrane. The MD simulations provide a molecular interpretation of the experiments and highlight that, in order to maximize the apolar interactions, the 3-O-derivative is embedded in the lipid hydrophobic region. This topographical position of the 3-O resveratrol analog perturbs the liquid-crystalline order of the lipid bilayer promoting membrane curvature and partial lipid loss from the vesicle. This finding reconciles with the lowering of the enthalpy of the lipid phase transition and the ability of the molecule to diffuse across membranes. The present data contribute to explain the different biological activity of the two molecules and evidence that membrane permeability is a key requirement for effective design of resveratrol derivatives to be used for therapeutic purposes. (C) 2012 Elsevier Inc. All rights reserved.

The hydrosoluble resveratrol derivative 3-O-phosphorylresveratrol was shown to be more cytotoxic against DU 145 prostate cancer cells than its analog 4'-O-phosphorylresveratrol. In an attempt to unveil the molecular determinants that lye at the root of their different biological effects, here we investigate the interactions of the two resveratrol derivatives with DMPC model membranes by using DSC, membrane permeation/poration assays and molecular dynamics. The results show that the 3-O-derivative interacts with DMPC membranes and diffuses across them. The 4'-O-derivative lies preferentially onto the surface of membrane. The MD simulations provide a molecular interpretation of the experiments and highlight that, in order to maximize the apolar interactions, the 3-O-derivative is embedded in the lipid hydrophobic region. This topographical position of the 3-O resveratrol analog perturbs the liquid-crystalline order of the lipid bilayer promoting membrane curvature and partial lipid loss from the vesicle. This finding reconciles with the lowering of the enthalpy of the lipid phase transition and the ability of the molecule to diffuse across membranes. The present data contribute to explain the different biological activity of the two molecules and evidence that membrane permeability is a key requirement for effective design of resveratrol derivatives to be used for therapeutic purposes

Human islet amyloid polypeptide (hIAPP) is known to misfold and aggregate into amyloid deposits that may be found in pancreatic tissues of patients affected by type 2 diabetes. Recent studies have shown that the highly amyloidogenic peptide LANFLVH, corresponding the N-terminal 12-18 region of IAPP, does not induce membrane damage. Here we assess the role played by the aromatic residue Phe in driving both amyloid formation and membrane interaction of LANFLVH. To this aim, a set of variant heptapeptides in which the aromatic residue Phe has been substituted with a Leu and Ala is studied. Differential scanning calorimetry (DSC) and membrane-leakage experiments demonstrated that Phe substitution noticeably affects the peptide-induced changes in the thermotropic properties of the lipid bilayer but not its membrane damaging potential. Atomic force microscopy (AFM), ThT fluorescence and Congo red birefringence assays evidenced that the Phe residue is not required for fibrillogenesis, but it can influence the self-assembling kinetics. Molecular dynamics simulations have paralleled the outcome of the experimental trials also providing informative details about the structure of the different peptide assemblies. These results support a general theory suggesting that aromatic residues, although capable of affecting the self-assembly kinetics of small peptides and peptide-membrane interactions, are not essential either for amyloid formation or membrane leakage, and indicate that other factors such as β-sheet propensity, size and hydrophobicity of the side chain act synergistically to determine peptide properties.

The massive computational resources available in the framework of a grid paradigm approach represent an emerging tool in the bioinformatics field. In this paper, we used the above approach in the rapid determination of the interactions between the ring-hydroxylating dioxygenase, comprised six enzymatic subunits, and polycyclic aromatic hydrocarbons (PAHs) in their optimal positions. The results were obtained by simulating enzyme dynamics at 300 K through molecular dynamics calculations. For the first time, the equilibrated structure of the dioxygenase revealed a network of channels throughout the enzyme that were sufficiently large to allow a flow of small ions or molecules from the inner core of the complex to its exterior surface. The ring-hydroxylating dioxygenase was able to interact with some of the studied PAHs. Additionally, not only the number of aromatic rings but also the PAH shape were critical in predicting the ability of the dioxygenase to interact with these types of molecules. Docking calculations shed light on a new possible binding site that is far from the enzymatic one, which is potentially interesting in considering the stability of the enzyme itself.

The intensive use of herbicides over the last few decades has caused a general increase of environmental pollution. It is thus very important to evaluate the possible genotoxic properties of these chemical compounds as well as identifying their mode of action. Phenylurea herbicides are selective agents widely used for the control of infestant plants. Of these herbicides, which are widely used in agriculture, we analysed four of the less intensively studied molecules. More precisely, we investigated the genotoxic effects of fenuron, chlorotoluron, diuron, and difenoxuron by analyses of chromosomal aberrations (CAs) and sister chromatid exchange (SCE) in exposed mammalian cells. We used the Chinese hamster ovary (CHO) and epithelial liver (CHEL) cell lines, endowed with the absence or the presence, respectively, of an enzymatic system to activate pro-mutagenic compounds. Our results show that all herbicides tested induce, at high concentrations, an increasing number of CAs in non-metabolising CHO cells. Instead, in the exposed CHEL cell line, the four herbicides induced CAs also at the lowest dose-level. In the CHEL cells, a statistically significant increase of SCE was also observed. The phenylurea herbicides showed direct genotoxic activity, but the cytogenetic effects were greatly enhanced after metabolic conversion. These data, together with other information on phenylurea herbicides, are of great interest from the environmental point of view, and for human health. In fact, intensive use of herbicides contaminates soil, surface water, groundwater and agricultural products, and thus should be taken in particular consideration not only for those initiatives to specifically protect exposed workers, but also to safeguard the health of consumers of agricultural products.

Islet amyloid polypeptide (IAPP) is a 37-residue hormone known to deposit as fibrillar aggregates in pancreatic β-cells of patients affected by T2DM. Although it has been proposed that both the fibrillogenic potential and membrane-activity may play a key role in IAPP cytotoxicity, a direct causative relationship between these two properties has not yet been firmly established. More recently, it has been observed that membrane damage may occur independently from fiber formation of IAPP and that these properties may be encoded by different sequences of IAPP. To further check this hypothesis, the membrane-activity and aggregation properties of the two neutral segments LANFLVH (IAPP12–18) and NNFGAIL (IAPP21–27), that recent theoretical studies have reported to possess the highest and the lowest fibrillogenic potential respectively, have been studied by means of a combined experimental and computational approach. The whole of the results demonstrate that if neutral peptides and lipids are employed, the most fibrillogenic peptide has the lowest membrane damaging effect and vice versa. These findings are expected to contribute to our rational understanding of the factors involved in the formation of amyloidosis and in the mechanisms of peptide-induced membrane damage.

Although the molecular determinants of Familial Amyotrophic Lateral Sclerosis (FALS) are still largely unknown, previous studies have demonstrated that aggregation of Cu, Zn superoxide dismutase (SOD1) mutants may play a causative role in FALS. It has been proposed that this pathogenic process occurs via a multi-step pathway involving metal loss, dimer dissociation and assembly of misfolded apo-monomers. The G37R, one of the many SOD1 mutations known to be associated to FALS, is difficult to be reconciled with this model because it is located far from the metal sites and the monomer-monomer interface. Consequently, an inspection of all the steps involved in G37R SOD1 misfolding is expected to provide hints in the understanding of the molecular basis of the disease. To this aim, an array of different computational strategies--i.e. Thermodynamic Integration (TI), implicit solvent Constant Temperature Molecular Dynamics (CTMD) and Steered Molecular Dynamics (SMD)--have been applied on the G37R SOD1 mutant. A comparison with parallel studies carried out for the Wild Type (WT) SOD1 pointed out that the mutation decreases the affinity of the protein for the Cu(ii) ion. Implicit solvents MD simulations performed on the two apo proteins revealed that in the mutant SOD1 a novel, stable H-bond network involving Arg37, Lys91, Lys36 and Leu38 is created thus confirming a pivotal role of this region in driving the biophysical properties of the entire protein. Finally, the presence of energetic "traps" in the force vs. elongation curves of G37R SOD1 is an indicator of the existence of intermediate states along the unfolding pathway which may lead to abnormal conformers. Our results support a general theory suggesting that the two major hypotheses regarding mutant SOD1 toxicity, i.e. aberrant copper redox chemistry and SOD1 misfolding are causally linked. In fact it is shown that the G37R mutation, although located far away the active site, may induce subtle modification in SOD1 leading to the loosening of metal binding and to the formation of metastable intermediate states along the unfolding pathway.

We studied the interaction of human amylin with dipalmitoylphosphatidylcholine and dipalmitoylphosphatidylserine vesicles using differential scanning calorimetry, circular dichroism and molecular modelling. The results suggest that inappropriate insertion of amylin into the hydrophobic core of a lipid bilayer could hinder the secretion of amylin from pancreatic beta-cells.

The role played by Ca(2+) ions in the interaction of the human islet amyloid polypeptide (hIAPP) with model membranes has been investigated by differential scanning calorimetry (DSC) and circular dichroism (CD) experiments. In particular, the interaction of hIAPP and its rat isoform (rIAPP) with zwitterionic dipalmitoyl-phosphatidylcholine (DPPC), negatively charged dipalmitoyl-phosphatidylserine (DPPS) vesicles and with a 3:1 mixtures of them, has been studied in the presence of Ca(2+) ions. The experiments have evidenced that amorphous, soluble hIAPP assemblies interact with the hydrophobic core of DPPC bilayers. Conversely, the presence of Ca(2+) ions is necessary to activate a preferential interaction of hIAPP with the hydrophobic core of DPPS membranes. These findings support the hypothesis that an impaired cellular homeostasis of Ca(2+) ions may promote the insertion of hIAPP into the hydrophobic core of carrier vesicles which is thought to contribute to an eventual intracellular accumulation of beta-sheet rich hIAPP aggregates.

Prion diseases are associated with an abnormal conformational transition involving the prion protein and are known to affect mammals. Here, the different mechanical behaviour of two mammalian, human (HuPrP) and Syrian hamster (ShaPrP), and two non-mammalian, chicken (ChPrP) and turtle (TuPrP), prions was assessed by steered molecular dynamics simulations performed on the globular domains of the four proteins. In mammalian prions a greater resistance to external stretching forces and an earlier occurrence of irreversible events were observed. The different unfolding profile of mammalian prions, ascribable to the intramolecular interactions involving helix 1 with helix 3, implicate the existence of metastable non-native states which may prompt abnormal pathways of protein misfolding and aggregation.

The role played by the alpha-helix in determining the structure, the stability and the unfolding mechanism of azurin was addressed by studying a helix-depleted azurin variant produced by site-directed mutagenesis. The protein structure was investigated by CD, 1D (1)H NMR, fluorescence spectroscopy measurements and MD simulations, whilst EPR, UV-visible and cyclic voltammetry experiments were carried out to investigate the geometry and the properties of the Cu(II) site. The effects of the alpha-helix depletion on the thermal stability and the unfolding pathway of the protein were determined by DSC, UV/visible and fluorescence measurements at increasing temperature. The results show that, in the absence of the alpha-helix segment, the overall protein structure is maintained, and that only the Cu site is slightly modified. In contrast, the protein stability is diminished by about 60% with respect to the wild-type azurin. Moreover, the unfolding pathway of the mutant azurin involves the presence of detectable intermediates. In comparison with previous studies concerning other small beta-sheet cupredoxins, the results as a whole support the hypothesis that the presence of the alpha-helix can switch the folding of azurin from a hierarchic to a nonhierarchic mechanism in which the highly conserved beta-sheet core provides a scaffold for cooperative folding of the wild-type protein.

International audience; A novel computational approach to the structural analysis of ordered beta-aggregation is presented and validated on three known amyloidogenic polypeptides. The strategy is based on the decomposition of the sequence into overlapping stretches and equilibrium implicit solvent molecular dynamics (MD) simulations of an oligomeric system for each stretch. The structural stability of the in-register parallel aggregates sampled in the implicit solvent runs is further evaluated using explicit water simulations for a subset of the stretches. The beta-aggregation propensity along the sequence of the Alzheimer's amyloid-beta peptide (Abeta(42)) is found to be highly heterogeneous with a maximum in the segment V(12)HHQKLVFFAE(22) and minima at S(8)G(9), G(25)S(26), G(29)A(30), and G(38)V(39), which are turn-like segments. The simulation results suggest that these sites may play a crucial role in determining the aggregation tendency and the fibrillar structure of Abeta(42). Similar findings are obtained for the human amylin, a 37-residue peptide that displays a maximal beta-aggregation propensity at Q(10)RLANFLVHSSNN(22) and two turn-like sites at G(24)A(25) and G(33)S(34). In the third application, the MD approach is used to identify beta-aggregation "hot-spots" within the N-terminal domain of the yeast prion Ure2p (Ure2p(1-94)) and to design a double-point mutant (Ure2p-N4748S(1-94)) with lower beta-aggregation propensity. The change in the aggregation propensity of Ure2p-N4748S(1-94) is verified in vitro using the thioflavin T binding assay.

The failure of the ubiquitin-proteasome system is involved in many diseases. Here, in order to assess the pH-induced changes in the physico-chemical properties of ubiquitin, DSC measurements have been carried out at 2.5 < pH < 7. Parallel 'in silico' pH titrations, suggest that there is a direct relationship between the calorimetric data and the electrostatic properties of ubiquitin. It is suggested that these pH-induced changes in the properties of ubiquitin may play a role in determining its ability to properly conjugate with its physiological targets.

DPPC dispersions containing DPPE with attached PEG of molecular masses 350, 2000 and 5000 were investigated by DSC in order to determine their phase behaviour and potential use as drug delivery systems. In comparison with previously obtained ESR data, DSC provided a definition of the lipid composition and temperature at which the vesicles are in a liquid crystalline phase. For DPPC DPPE-PEG 350 the composition range is at molar fractions 0

Free energy perturbation/molecular dynamics simulations have been carried out on copper/azurin systems calculating the binding affinities of copper (II) ion to azurin either in the native or in the unfolded state. In order to test the validity of the strategy adopted for the calculations and to establish what force field is suitable for these kinds of calculations, three different force fields, AMBER, CVFF, and CFF, have been alternatively used for the calculations and the results have been compared with experimental data obtained by spectroscopic titrations of copper (II)/azurin solutions and denaturation experiments. Our findings have pointed out that only CFF gives satisfactory results, thus providing a reliable tool for copper binding simulations in copper protein. © 2003 Wiley Periodicals, Inc. J Comput Chem 6: 779–785, 2003

4 This corrects the caption to Figure 1. It should read as follows: 5 Comparison of the MAO A (PDB ID: 2Z5X, blue) active 6 site cavity with that of MAO B-TCP-2-BFI (PDB ID: 2XCG, 7 pink). The FAD cofactors are shown in line form. The residues 8 in contact with 2-BFI (cyan) are displayed in stick format: 9 Pro102, Ile199, Gln206, Ile316, and Tyr326 in MAO B (red) 10 with the equivalent residues Ala111, Phe208, Gln215, Ile325, 11 and Ile335 in MAO A (blue). Residues 109−112 of the loop at 12 the entrance to the active site cavity in MAO A are shown in 13 blue stick format. Erratum