The role of aromatic side-chains in amyloid growth and membrane interaction of the islet amyloid polypeptide fragment LANFLVH.

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 ß-sheet propensity, size and hydrophobicity of the side chain act synergistically to determine peptide properties.

Unveiling the unfolding pathway of FALS associated G37R SOD1 mutant: a computational study.

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.

Calcium-activated membrane interaction of the islet amyloid polypeptide: implications in the pathogenesis of type II diabetes mellitus.

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.

The role played by the alpha-helix in the unfolding pathway and stability of azurin: switching between hierarchic and nonhierarchic folding.

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.

Thermodynamic analysis of the contributions of the copper ion and the disulfide bridge to azurin stability: synergism among multiple depletions.

The stabilizing potential of the copper ion and the disulfide bridge in azurin has been explored with the aim of inspecting the ways in which these two factors influence one another. Specifically, whether copper and disulfide contributions to protein stability are additive has been examined. To this aim, the thermal unfolding of a copper-depleted mutant lacking the disulfide bridge between Cys3 and Cys26 (apo C3A/C26A azurin) was studied by differential scanning calorimetry. A comparison of the unfolding parameters of holo and apo C3A/C26A azurin with the apo C3A/C26A protein has shown that the effects of simultaneous copper and disulfide depletion are additive only at two temperatures: T=15 degrees C and T=67 degrees C. Within this range the presence of the copper ion and the disulfide bridge has a positive synergistic effect on azurin stability. These findings might have implications for the rational use of the stabilizing potential of copper and disulfides in copper protein engineering.

Free energy perturbation and molecular dynamics calculations of copper binding to azurin.

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.

A model for the thermal unfolding of amicyanin.

In the present study the thermal unfolding of amicyanin has been addressed using differential scanning calorimetry, fluorescence emission, optical density, circular dichroism and electron paramagnetic resonance. The combined use of these techniques has allowed us to assess, during unfolding of the protein, its global conformational changes in relationship to the local structural modifications occurring in the copper environment and close to the fluorescent chromophore Trp46 of the protein. The thermal transition from the native to the denatured state is on the whole irreversible and occurs in the temperature range between 65 and 72 degrees C, depending on the scan rate and technique used. Amicyanin as a whole shows a complex unfolding pathway, which has been described in terms of a three-step model: N <--> U --> F1 --> F2. According to this model, in the first step the native state of the protein (N) goes reversibly to the unfolded state (U), in the second one U goes irreversibly to F1 and, finally, the state F2 is irreversibly reached in the third step. Kinetic factors prevent the experimental separation of these steps. Nevertheless, the comparison of the data obtained with the different experimental techniques testifies the presence, within the unfolding pathway, of some intermediate states, although not sufficiently long-lived to allow a detailed characterization. A first intermediate transient state has been identified around 68 degrees C, whereas a second one can be related to conformational changes that involve the copper environment. Finally, an exothermal phenomenon, caused by irreversible rearrangements of the melted polypeptide chains, is evidenced. In addition, according to the EPR findings, the type 1 copper ion, which is four-fold coordinated by two N and two S atoms in a distorted tetrahedron in the native state of the protein, shows type 2 features after denaturation. A mathematical model simulating the unfolding Cp(exc) profile has been also developed.