Role of dietary antioxidant (-)-epicatechin in the development of ß-lactoglobulin fibrils.

Under specific physico-chemical conditions ß-lactoglobulin is seen to form fibrils structurally highly similar to those that are formed by the amyloid-like proteins associated with neurodegenerative disorders, such as Alzheimer and Parkinson diseases. In the present study we provide insights on the possible role that the dietary flavonoid (-)-epicatechin plays on ß-lactoglobulin fibril formation. Fibril formation is induced by keeping ß-lactoglobulin solutions at pH2.0 and at a temperature of 80°C for 24h. Atomic Force Microscopy measurements suggest that, by adding (-)-epicatechin in the solution, fibrils density is visibly lowered. This last observation is confirmed by Fluorescence Correlation Spectroscopy experiments. With the use of Fourier Transform IR spectroscopy we monitored the relative abundances of the secondary structures components during the heating process. We observed that in the presence of (-)-epicatechin the spectral-weight exchange between different secondary structures is partially inhibited. Molecular Dynamics simulations have been able to provide an atomistic explanation of this experimental observation, showing that (-)-epicatechin interacts with ß-lactoglobulin mainly via the residues that, normally in the absence of (-)-epicatechin, are involved in ß-sheet formation. Unveiling this molecular mechanism is an important step in the process of identifying suitable molecules apt at finely tuning fibril formation like it is desirable to do in food industry applications.

A first-principle calculation of the XANES spectrum of Cu(2+) in water.

The progress in high performance computing we are witnessing today offers the possibility of accurate electron density calculations of systems in realistic physico-chemical conditions. In this paper, we present a strategy aimed at performing a first-principle computation of the low energy part of the X-ray Absorption Spectroscopy (XAS) spectrum based on the density functional theory calculation of the electronic potential. To test its effectiveness, we apply the method to the computation of the X-ray absorption near edge structure part of the XAS spectrum in the paradigmatic, but simple case of Cu(2+) in water. In order to keep into account the effect of the metal site structure fluctuations in determining the experimental signal, the theoretical spectrum is evaluated as the average over the computed spectra of a statistically significant number of simulated metal site configurations. The comparison of experimental data with theoretical calculations suggests that Cu(2+) lives preferentially in a square-pyramidal geometry. The remarkable success of this approach in the interpretation of XAS data makes us optimistic about the possibility of extending the computational strategy we have outlined to the more interesting case of molecules of biological relevance bound to transition metal ions.

The role of Zn ions in the interaction between SARS-CoV-2 orf7a protein and BST2/tetherin.

In this paper, we provide evidence that Zn 2 + ions play a role in the SARS-CoV-2 virus strategy to escape the immune response mediated by the BST2-tetherin host protein. This conclusion is based on sequence analysis and molecular dynamics simulations as well as X-ray absorption experiments [1].

Development of a molecularly imprinted polymer-matrix solid-phase dispersion method for selective determination of ß-estradiol as anabolic growth promoter in goat milk.

A simple, fast, and sensitive method for determination of 17 ß-estradiol (E2) in goat milk samples has been developed by combining selective molecularly imprinted matrix solid-phase dispersion (MIP-MSPD) and liquid chromatography with diode-array detection (DAD). The molecularly imprinted polymer was synthesized by use of 17ß-estradiol as template molecule, methacrylic acid as functional monomer, ethylene glycol dimethacrylate as crosslinker monomer, azobisisobutyronitrile as initiator, and acetonitrile as porogen, and was used as selective solid support for matrix solid-phase dispersion. The selected dispersant had high affinity for E2 in the goat milk matrix and the extract obtained was sufficiently clean for direct injection for HPLC analysis without any interferences from the matrix. The proposed MIP-MSPD method was validated for linearity, precision, accuracy, decision limit (CCα) and detection capability (CCß), in accordance with European Commission Decision 2002/657/EC criteria. Linearity ranged from 0.3-10 µg g(-1) (correlation coefficient r(2) > 0.999). Mean recovery of E2 from goat milk samples at different spiked levels was between 89.5 and 92.2%, with RSD values within 1.3-2%. CCα and CCß values were 0.36 and 0.39 µg g(-1), respectively. The developed MIP-MSPD method was successfully applied to direct determination of E2 in goat milk samples.

Metal Ion Binding in Wild-Type and Mutated Frataxin: A Stability Study.

This work studies the stability of wild-type frataxin and some of its variants found in cancer tissues upon Co2+ binding. Although the physiologically involved metal ion in the frataxin enzymatic activity is Fe2+, as it is customarily done, Co2+ is most often used in experiments because Fe2+ is extremely unstable owing to the fast oxidation reaction Fe2+ → Fe3+. Protein stability is monitored following the conformational changes induced by Co2+ binding as measured by circular dichroism, fluorescence spectroscopy, and melting temperature measurements. The stability ranking among the wild-type frataxin and its variants obtained in this way is confirmed by a detailed comparative analysis of the XAS spectra of the metal-protein complex at the Co K-edge. In particular, a fit to the EXAFS region of the spectrum allows positively identifying the frataxin acidic ridge as the most likely location of the metal-binding sites. Furthermore, we can explain the surprising feature emerging from a detailed analysis of the XANES region of the spectrum, showing that the longer 81-210 frataxin fragment has a smaller propensity for Co2+ binding than the shorter 90-210 one. This fact is explained by the peculiar role of the N-terminal disordered tail in modulating the protein ability to interact with the metal.

The role of metals in beta-amyloid peptide aggregation: X-Ray spectroscopy and numerical simulations.

The aim of this review is to show how the challenging problem of understanding the physico-chemical basis of protein misfolding and aggregation which are at the origin of plaque formation in amyloid pathologies can be successfully investigated with a combination of modern spectroscopic techniques and advanced first principle numerical simulations. Within the vast group of diseases (more than 20) characterized by extra-cellular deposition of fibrillar material and generically called Amyloidosis, we shall focus on the Alzheimer's disease, a progressive and devastating neurodegenerative pathology affecting an important fraction of the world aged population. Well identified peptides (the so called Abeta-peptides) undergo a misfolding process during the development of the disease. An important, but not yet fully elucidated, rôle appears to be played in these processes by transition metals (mainly copper and zinc) that have been observed to be present in large amounts in patient's neurological plaques. Starting from this observation, a number of interesting results concerning the structural properties of the relevant metal-peptide binding site, emerging from the interplay between X-ray Absorption Spectroscopy experiments, and ab initio molecular dynamics simulations of the Car-Parrinello type will be reported and discussed.

Autonomic and behavioural thermoregulation in tennis.

OBJECTIVES: This report describes physiological and behavioural mechanisms behind the control of body temperature and thermal comfort during competitive singles tennis. METHODS: Thermoregulatory responses and workload were observed during "best of three sets" tennis matches among 25 players. In total, 94 matches were played in ambient temperatures ranging from 14.5 to 38.4 degrees C. The thermal environment was assessed by dry bulb, wet bulb and natural wet bulb temperatures, globe temperature and wind speed. Core body and skin temperatures were recorded each minute throughout the match, and body mass and fluid intake were measured before the match, after 30 minutes of play and at the completion of the match to determine sweat rate. Subjective ratings of thermal strain included thermal comfort, sweatiness and perceived exertion. Workload observations included match, game and point durations, and the proportion of match time spent in play (effective playing time). RESULTS: Change in rectal temperature was positively correlated with point duration (p<0.001) and effective playing time (p<0.05). Sweat rate showed positive associations with air (p<0.0001), rectal (p<0.03) and skin (p<0.0001) temperature. Thermal comfort was reduced with increasing rectal (p<0.03) and skin (p<0.0001) temperature. Point duration and effective playing time were reduced when conditions were rated increasingly difficult (p<0.002 and p<0.0002, respectively). CONCLUSION: Autonomic (increase in sweat rate) and behavioural (reduction in workload) thermoregulation are responsible for the control of body temperature and thermal comfort during tennis.

Thermoregulatory responses during competitive singles tennis.

OBJECTIVES: To provide examples of thermoregulatory responses during competitive singles tennis and comparisons with continuous, steady-state running. METHODS: Typical examples of body core (rectal) temperature, skin temperature and heart rate were selected to show the differing characteristics of tennis and running, and the corresponding thermal environments. Rectal and skin temperatures were logged each minute and heart rate logged every 15 seconds throughout the competitive "best of three sets" singles tennis matches and 60-minute continuous, steady-state running trials. Tennis matches were completed outdoors in widely varying thermal environments, and the running trials were completed in the laboratory under stable conditions. RESULTS: Rectal temperature in tennis was raised only slightly above resting levels, reaching a plateau relative to the exercise intensity. Rectal temperature during tennis was found to take longer to reach a plateau than continuous, steady-state exercise. Skin temperature during tennis varied widely depending on environmental air temperature, and was lower than that of runners at the same air temperature. Heart rate was very similar between opponents for both average and response characteristics during tennis. A wider range and higher peak values were found for tennis players compared with runners. CONCLUSIONS: This report provides a descriptive account of thermoregulatory response characteristics during singles tennis. Differences between outdoor tennis and continuous, steady-state running in the laboratory for each of these responses were found.

Designing effective anticancer-radiopeptides. A Molecular Dynamics study of their interaction with model tumor and healthy cell membranes.

One of the greatest merit of the use of radiopeptides in oncology is their selectivity which, however, brings about the drawback that each radiopeptide is specific for a given tumor type. To overcome this problem the direction currently taken in drug design is that of radiolabelling peptide hormones (or their analogues), relying on their intrinsic ability to bind to specific receptors in precise areas of the human body, at the cost, however, of a poor selectivity against healthy cells. We present here an extensive Molecular Dynamics study of a promising alternative inspired by the mechanism through which antimicrobial peptides interact with the negatively charged bacterial membranes. Appropriately modifying the human antimicrobial peptide, LL-37, we designed a functionalized radionuclide carrier capable of binding more strongly to the negatively charged (model) tumor membranes than to the neutral healthy ones. The mechanism behind this behaviour relies on the fact that at the slight acidic pH surrounding tumor tissues the histidines belonging to the peptide get protonated thus making it positively charged. We have investigated by an extended numerical study the way in which this artificial peptide interacts with models of tumor and healthy cell membranes, proving by Potential Mean Force calculations that the affinity of the peptide to model tumor membranes is significantly larger than to healthy ones. These features (high affinity and generic tumor selectivity) recommend antimicrobial derived customized carriers as promising theranostic constructs in cancer diagnostic and therapy.

Human insulin fibrillogenesis in the presence of epigallocatechin gallate and melatonin: Structural insights from a biophysical approach.

Fibrillogenesis of monomeric human insulin in the presence or absence of (-)-epigallocatechin-3-gallate and melatonin was here investigated using a multi-technique approach. Results from Raman and Infrared spectroscopy pointed out that a high content of intermolecular ß-sheet aggregates is formed after long-term incubation. However, near UV experiments, Dynamic Light Scattering, Thioflavin-T fluorescence measurements and Atomic Force Microscopy revealed that the kinetics from native-to-fibrillar state of insulin is hampered only in the presence of (-)-epigallocatechin-3-gallate. Molecular dynamic simulations indicated that this compound binds near the B11-B18 protein segment, where hydrophobic residues responsible for the beginning of cooperative aggregation are located. Such a preferential binding region is not recognized by melatonin, a highly mobile molecule, which indeed does not affect fibril formation. The results of the present study demonstrate that (-)-epigallocatechin-3-gallate interferes with the insulin nucleation phase, giving rise to amorphous aggregates in the early stages of the aggregation process.

The effect of ß-sheet breaker peptides on metal associated Amyloid-ß peptide aggregation process.

Far-UV Circular Dichroism experiments and Atomic Force Microscopy tomography are employed to assess the impact of ß-sheet breakers on the Aß1-40 peptide aggregation process in the presence of Cu2+ or Zn2+ transition metals. In this work we focus on two specific 5-amino acids long ß-sheet breakers, namely the LPFFD Soto peptide, already known in the literature, and the LPFFN peptide recently designed and studied by our team. We provide evidence that both ß-sheet breakers are effective in reducing the Aß1-40 aggregation propensity, even in the presence of metal ions.

Nucleation of the iron core occurs at the three-fold channels of horse spleen apoferritin: an EXAFS study on the native and chemically-modified protein.

Extended X-ray absorbance fine structure measurements have been carried out on the initial Fe(III)-apoferritin complex at a Fe/subunit ratio of 2 in native and modified horse spleen apoferritin. Analysis of the data indicates that in the native protein the iron forms a protein-bound polynuclear cluster (Fe-Fe distance 3.4 A) with a first coordination sphere constituted by 5-6 low-Z atoms, e.g., nitrogen atoms, carboxylate-like ligands or oxo bridges between the iron atoms. Modification of Cys-126, a residue localized on the outer surface of the hydrophilic three-fold channels, with p-chloromercuribenzoate (PMB) or phenylmercuric acetate (PMA) brings about distinctive differences. In particular, in the PMB-reacted protein the feature assigned to the iron-iron interaction disappears from the spectrum, whilst in the PMA-reacted protein the main differences with respect to the native protein are observed at the level of the first coordination sphere. These results confirm the formation of protein-Fe(III)-clusters and localize these sites at the hydrophilic three-fold channels of horse spleen apoferritin.

Is the activity-linked electrostatic gradient of bovine Cu, Zn superoxide dismutases conserved in homologous enzymes irrespective of the number and distribution of charges?

Electrostatic potential calculations have been performed on three different Cu, Zn superoxide dismutases (superoxide: superoxide oxidoreductase, EC 1.15. 1.1), in order to evaluate the degree of conservation of the pattern of electrostatic interactions between O2- and the active site recently pointed out in bovine Cu Zn SOD. The three Cu, Zn SODs that have been selected for this study, namely the bovine, ovine, and porcine enzymes, are highly homologous as to reasonably assume identical three-dimensional structure but display large differences in their net charge, as shown by their pI's, which span over a wide pH range: 8.0 (sheep), 6.5 (pig), 5.2 (ox). Despite such a large difference in the net protein charge and in the spatial arrangement of electrostatic charges, electrostatic potential calculations show that the electrostatic channel directing the negatively charged substrate toward the positive catalytic site is strictly preserved with the same features for the three proteins. This suggests that the electrostatic funnel for conducting small anions into the active site is a highly conservative property in the evolution of Cu, Zn SOD.

The role of Zn in the interplay among Langmuir-Blodgett multilayer and myelin basic protein: a quantitative analysis of XANES spectra.

We have performed a quantitative analysis of the X-ray absorption near-edge structure (XANES) spectra at the Zinc K-edge of systems formed by phospholipid Langmuir-Blodgett multilayers (LBMLs) in the presence and in the absence of myelin basic protein (MBP) and in two hydration conditions. These spectra have been analysed by a new procedure called Minuit XANes (MXAN) which is able to perform a quantitative fit of XANES data in terms of structural parameters. By this method, we have been able to correlate the relevant differences between the spectra observed in the XANES range with the coordination changes due to reduction of the space around the Zinc when the level of hydration is lowered and/or the myelin basic protein is added. These spectral differences are peculiar of the XANES energy range, and are not present in the extended X-ray absorption fine structure (EXAFS) energy range where the analysis was previously performed. With this investigation, we give an unambiguous answer to the question of the role of zinc in such complexes by showing that the metal interacts with both the phospholipid heads of the substrate and the myelin basic protein.

Tetanus and botulism neurotoxins: a novel group of zinc-endopeptidases.

Tetanus and botulinum neurotoxins are produced by bacteria of the genus Clostridium and cause the paralytic syndromes of tetanus and botulism with a persistent inhibition of neurotransmitter release at central and peripheral synapses, respectively. These neurotoxins consist of two disulfide-linked polypeptides: H (100 kDa) is responsible for neurospecific binding and cell penetration of L(50 kDa), a zinc-endopeptidase specific for three protein subunits of the neuroexocytosis apparatus. Tetanus neurotoxin and botulinum neurotoxins serotypes B, D, F, and G cleave at single sites, which differ for each neurotoxin. VAMP/synaptobrevin, a membrane protein of the synaptic vesicles. Botulinum A and E neurotoxins cleave SNAP-25, a protein of the presynaptic membrane, at two different carboxyl-terminal peptide bonds. Serotype C cleaves specifically syntaxin, another protein of the nerve plasmalemma. The target specificity of these metallo-proteinases relies on a double recognition of their substrates based on interactions with the cleavage site and with a non contiguous segment that contains a structural motif common to VAMP, SNAP-25 and syntaxin.

A micro-environmental study of the Zn(+2)-Aß1-16 structural properties.

Relying on a combination of classical molecular dynamics and hybrid QM/MM computational methods, we study the influence of the nature of the local physico-chemical environment on the structural features of ß-amyloid peptides complexed with Zn(+2) ions. The analysis is carried out by comparing among themselves different Zn(+2)-ligand force fields and studying their influence on metal coordination and long-range peptide folding. The system in the non-physiological so-called "gas phase" (no solvent) was also simulated with the purpose of identifying to what extent, if at all, the solvent can affect the Zn coordination mode, besides its long-range structural properties. There are two main results of this investigation. The first is that the Zn(+2) coordination mode in classical molecular dynamics simulations markedly depends on the partial charge attributed to the ion and the atoms surrounding it. Comparing with experiments, it is possible to identify the most appropriate Zn(+2) force field for the Zn(+2)-Aß1-16 complex in study. Secondly, although the presence of water naturally influences the peptide folding propensity, it does not affect the structure of the Zn(+2) inner coordination shell. A useful way to validate classical results and in particular those referring to the structural differences visible when different force fields are employed, was to use a hybrid QM/MM optimization step. When the classical system configurations are submitted to such a quantum minimization step, the geometries of the resulting Zn(+2) site turn out to be all very similar and structurally in good agreement with what is experimentally known.

Studying the Cu binding sites in the PrP N-terminal region: a test case for ab initio simulations.

First principle ab initio molecular dynamics simulations of the Car-Parrinello type have proved to be of invaluable help in understanding the microscopic mechanisms of chemical bonding both in solid state physics and in structural biophysics. In this work we present as a test case a study of the Cu coordination mode at the Prion Protein binding sites localized in the N-terminal octarepeat region. Using medium size PC-clusters, we are able to deal with systems with up to about 350 atoms and 10(3) electrons for as long as approximately 2 ps. With a foreseeable forthcoming scaling up of the available CPU times by a factor 10(3), one can hope to be soon able to simulate systems of biological interest of realistic size and for physical times of the order of the nanosecond.

The stress tensor of a molecular system: an exercise in statistical mechanics.

We prove that conservation of the stress tensor is a consequence of the invariance of the partition function under canonical diffeomorphisms. From this observation a simple and general derivation of the formula which gives the local expression of the stress tensor of a molecular system in terms of its microscopic degrees of freedom readily follows. The derivation is valid in the canonical as well as the microcanonical ensemble. It works both in the classical and in the quantum mechanical settings and for arbitrary boundary conditions. In particular, if periodic boundary conditions are assigned to the system, the usual minimal-image prescription is naturally born out for mathematical consistency. An interesting outcome of our general analysis is that only in the case of a short-range interaction potential a truly local formula for the stress tensor can exist.