Adriana Pietropaolo

Molecular Mechanism of Polyacrylate Helix Sense Switching across Its Free Energy Landscape

Helical polymers with switchable screw sense are versatile frameworks for chiral functional materials. In this work, we reconstructed the free energy landscape of helical poly(2,7-bis(4-tert-butylphenyl)fluoren-9-yl acrylate) [poly(BBPFA)], as its racemization is selectively driven by light without any rearrangement of chemical bonds. The chirality inversion was enforced by atomistic free energy simulations using chirality indices as reaction coordinates. The free energy landscape reproduced the experimental electronic circular dichroism spectra. We propose that the chirality inversion of poly(BBPFA) proceeds from a left-handed 31 helix via multistate free energy pathways to reach the right-handed 31 helix. The inversion is triggered by the rotation of biphenyl units with an activation barrier of 38 kcal/mol. To the best of our knowledge, this is the first report on the chiral inversion mechanism of a helical polymer determined in a quantitative way in the framework of atomistic free energy simulations.

Copper(II) complexes with chicken prion repeats: influence of proline and tyrosine residues on the coordination features.

The prion protein (PrPc) is a copper-binding glycoprotein that can misfold into a β-sheet-rich and pathogenic isoform (PrPsc) leading to prion diseases. The first non-mammalian PrPc was identified in chicken and it was found to keep many structural motifs present in mammalian PrPc, despite the low sequence identity (approximately 40%) between the two primary structures. The present paper describes the synthesis and the coordination properties of some hexapeptide fragments (namely, PHNPGY , HNPGYP and NPGYPH) as well as a bishexapeptide (PHNPGYPHNPGY), which encompasses two hexarepeats. The copper(II) complexes were characterized by means of potentiometric, UV–vis, circular dichroism and electron paramagnetic resonance techniques. We also report the synthesis of three hexapeptides (PHNPGF, HNPGFP and NPGFPH), in which one tyrosine was replaced by phenylalanine as well as two bishexapeptides in which either one (PHNPGFPHNPGY and PHNPGYPHNPGF), or two tyrosines were replaced by phenylalanine, in order to check whether tyrosine was involved in copper(II) binding. Overall, the results indicate that the major copper(II) species formed by the chicken PrP dodecapeptides are stabler than the analogous species reported for the peptide fragments containing two octarepeat peptides from the mammalian prion protein. It is concluded that the presence of four prolyl residues, that are break points in copper coordination, induces the metal-assisted formation of macrochelates as well as the formation of binuclear species. Furthermore, it has been shown that the phenolic group is directly involved in the formation of copper binuclear species.

Predicting the Switchable Screw Sense in Fluorene-Based Polymers†

A chirality-switching free-energy landscape was reconstructed on a 43-mer of poly(9,9-dioctylfluoren-2,7-diyl) (PDOF). The simulations were conducted on amorphous silica surface as well as in the vacuum phase for a single chain or for a group of sixteen chains. The achiral-to-chiral transition occurs only on amorphous silica (activation free-energy 35 kcal mol(-1) ), where the enantiomeric (homochiral) basins are detected. This was supported by the experiments where effective chirality induction to PDOF using circularly polarized light (CPL) was attained only for a film deposited on a quartz glass and not for a solution or a suspension. These results indicate that interactions of PDOF with amorphous silica play a crucial role in chirality switching. Importance of chain assembling was also indicated. Theoretical ECD spectra of the enantiomeric basins containing a 51 helix reproduce the experimental spectra.

Metal ions affect insulin-degrading enzyme activity

Insulin degradation is a finely tuned process that plays a major role in controlling insulin action and most evidence supports IDE (insulin-degrading enzyme) as the primary degradative agent. However, the biomolecular mechanisms involved in the interaction between IDE and its substrates are often obscure, rendering the specific enzyme activity quite difficult to target. On the other hand, biometals, such as copper, aluminum and zinc, have an important role in pathological conditions such as Alzheimers disease or diabetes mellitus. The metabolic disorders connected with the latter lead to some metallostasis alterations in the human body and many studies point at a high level of interdependence between diabetes and several cations. We have previously reported (Grasso et al., Chem. Eur. J. 17 (2011) 2752-2762) that IDE activity toward Aβ peptides can be modulated by metal ions. Here, we have investigated the effects of different metal ions on the IDE proteolytic activity toward insulin as well as a designed peptide comprising a portion of the insulin B chain (B20-30), which has a very low affinity for metal ions. The results obtained by different experimental techniques clearly show that IDE is irreversibly inhibited by copper(I) but is still able to process its substrates when it is bound to copper(II).

Copper, BDNF and its N-terminal Domain: Inorganic Features and Biological Perspectives

Brain-derived neurotrophic factor (BDNF) is a neurotrophin that influences development, maintenance, survival, and synaptic plasticity of central and peripheral nervous systems. Altered BDNF signaling is involved in several neurodegenerative disorders including Alzheimers disease. Metal ions may influence the BDNF activity and it is well known that the alteration of Cu(2+) homeostasis is a prominent factor in the development of neurological pathologies. The N-terminal domain of BDNF represents the recognition site of its specific receptor TrkB, and metal ions interaction with this protein domain may influence the protein/receptor interaction. In spite of this, no data inherent the interaction of BDNF with Cu(2+) ions has been reported up to now. Cu(2+) complexes of the peptide fragment BDNF(1-12) encompassing the sequence 1-12 of N-terminal domain of human BDNF protein were characterized by means of potentiometry, spectroscopic methods (UV/Vis, CD, EPR), parallel tempering simulations and DFT-geometry optimizations. Coordination features of the acetylated form, Ac-BDNF(1-12), were also characterized to understand the involvement of the terminal amino group. Whereas, an analogous peptide, BDNF(1-12)D3N, in which the aspartate residue was substituted by an asparagine, was synthesized to provide evidence on the possible role of carboxylate group in Cu(2+) coordination. The results demonstrated that the amino group is involved in metal binding and the metal coordination environment of the predominant complex species at physiological pH consisted of one amino group, two amide nitrogen atoms, and one carboxylate group. Noteworthy, a strong decrease of the proliferative activity of both BDNF(1-12) and the whole protein on a SHSY5Y neuroblastoma cell line was found after treatment in the presence of Cu(2+). The effect of metal addition is opposite to that observed for the analogous fragment of nerve growth factor (NGF) protein, highlighting the role of specific domains, and suggesting that Cu(2+) may drive different pathways for the BDNF and NGF in physiological as well as pathological conditions.

A chirality index for investigating protein secondary structures and their time evolution

We propose a methodology for the description of the secondary structure of proteins, based on assigning a chirality parameter to short aminoacid sequences according to their arrangement in space at a certain time. We validated the method on ideal and crystalline structures, showing that it can assign secondary structures and that this assignment is robust with respect to random conformational perturbations. From the values of the index and its pattern along a sequence it is possible to recognize many structural motifs of a protein, and in particular poly‐L‐proline II left‐handed helices, often not detected by secondary structure assignment algorithms.

Unusual Cyclodextrin Derivatives as a New Avenue to Modulate Self‐ and Metal‐Induced Aβ Aggregation

Mounting evidence suggests an important role of cyclodextrins in providing protection in neurodegenerative disorders. Metal dyshomeostasis is reported to be a pathogenic factor in neurodegeneration because it could be responsible for damage involving oxidative stress and protein aggregation. As such, metal ions represent an effective target. To improve the metal-binding ability of cyclodextrin, we synthesized three new 8-hydroxyquinoline-cyclodextrin conjugates with difunctionalized cyclodextrins. In particular, the 3-difunctionalized regioisomer represents the first example of cyclodextrin with two pendants at the secondary rim, resulting in a promising compound. The derivatives have significant antioxidant capacity and the powerful activity in inhibiting self-induced amyloid-β aggregation seems to be led by synergistic effects of both cyclodextrin and hydroxyquinoline. Moreover, the derivatives are also able to complex metal ions and to inhibit metal-induced protein aggregation. Therefore, these compounds could have potential as therapeutic agents in diseases related to protein aggregation and metal dyshomeostasis.

Copper(II) complexes with an avian prion N-terminal region and their potential SOD-like activity

Potentiometric and spectroscopic (UV-Vis, CD and EPR) studies were carried out on copper(II) complexes with chicken prion protein N-terminal fragments, Ac-(PHNPGY)(4)-NH(2), and the mutated residue, Ac-(PHNPGF)(4)-NH(2), to assess the role of tyrosine in the copper coordination. Both thermodynamic and spectroscopic results indicate that chicken prion fragments are not able to bind more than two copper ions and only with the involvement of side chain tyrosine groups. The prevailing complex shows one copper ion bound to four imidazole nitrogen atoms in the 1:1 metal to ligand ratio systems. The superoxide dismutase (SOD)-like activity of copper(II) complexes with the avian peptides and mammal analogue, Ac-(PHGGGWGQ)(4)-NH(2), was also investigated by means of Pulse radiolysis. The copper(II) complexes with avian peptides do not display SOD-like activity, while very low activity has been detected for the copper(II) complexes with mammalian tetraoctarepeat.

The inorganic perspectives of neurotrophins and Alzheimer's disease

The recent metal hypothesis represents an attempt of a new interpretation key of Alzheimers disease (AD) to overcome the limits of amyloid cascade. Neurons need to maintain metal ions within a narrow range of concentrations to avoid a detrimental alteration of their homeostasis, guaranteed by a network of specific metal ion transporters and chaperones. Indeed, it is well known that transition metal ions take part in neuromodulation/neurotrasmission. In addition, they are prominent factors in the development and exacerbation of neurodegeneration. Neurotrophins are proteins involved in development, maintenance, survival and synaptic plasticity of central and peripheral nervous systems. A neurotrophin hypothesis of AD has been proposed, whereas the link between neurotrophic factor, the amyloid cascade and biometals has not been taken into account. As a matter of fact, there is a significant overlap between brain areas featured by metal ion dys-homeostasis, and those where the neurotrophins exert their biological activity. Metal ions can directly modulate their activities, through conformational changes, and/or indirectly by activating their downstream signaling in a neurotrophin-independent mode. The focus of this review is on the molecular aspects of Zn(2+) and Cu(2+) interactions with neurotrophins, with the aim to shed light on the intricate mechanisms involving metallostasis and proteostasis in AD.

Intramolecular Weak Interactions in the Thermodynamic Stereoselectivity of Copper(II) Complexes with Carnosine–Trehalose Conjugates

The interactions of metal ions with chiral molecules are of particular interest for relevant biochemical processes, as many of them are made possible only with a selected chirality of the stereocenters. In this work we report a study of the stereoselectivity of copper(II) complexes with D-trehalose-L-carnosine and D-trehalose-D-carnosine as a prototypical case of natural chirality selection. The interest in L-carnosine dipeptide is compounded by its antioxidant and antitumor properties, which are further enhanced when combined with D-trehalose. Potentiometric, calorimetric, and UV/circular dichroism (CD) spectroscopic measurements show that the copper(II) dimer of D-trehalose-L-carnosine is more stable than the D-trehalose-D-carnosine diastereoisomeric copper(II) dimer (log β(L)(22-2) - log β(D)(22-2) = 3.6). Free-energy calculations highlight that the cause of this different behavior lies with different intramolecular weak interactions between the diastereoisomers. The different pattern of hydrogen bonds and the different CH-π interactions between the π-electron-rich imidazole and the α-glucose rings are more favorable by 5 kcal mol(-1) in the L dimer.