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.

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.

Coarsening effect on island-size scaling: the model case InAs/GaAs(001).

The distributions of the size of islands and of the capture zones are discussed comparatively, both experimentally and numerically, for the case of a sudden nucleation process with and without coarsening. The experiments were performed by growing InAs islands on GaAs(001) and the coarsening was altered by varying the temperature. In the two-dimensional kinetic Monte Carlo simulations a single-species diffusing adatom was taken into account, and the coarsening was altered in this case by modifying the binding energy between adatoms and islands. The results show that size and capture zone distributions overlap only when coarsening can be disregarded.

Elastic incoherent neutron scattering as a probe of high pressure induced changes in protein flexibility.

We report here the results of elastic incoherent neutron scattering experiments on three globular proteins (trypsin, lysozyme and beta-lactoglobulin) in different pressure intervals ranging from 1 bar to 5.5 kbar. A decrease of the mean square hydrogen fluctuations, u(2), has been observed upon increasing pressure. Trypsin and beta-lactoglobulin behave similarly while lysozyme shows much larger changes in u(2). This can be related to different steps in the denaturing processes and to the high propensity of lysozyme to form amyloids. Elastic incoherent neutron scattering has proven to be an effective microscopic technique for the investigation of pressure induced changes in protein flexibility.

Signatures of the hydrogen bonding in the infrared bands of water.

Infrared spectroscopy measurements have been completed over a wide range of frequencies allowing to measure the evolution of both intramolecular and intermolecular vibrational modes in water as a function of temperature. Emphasis is made on the high frequency OH stretching band and the so-called connectivity band that lies in the far infrared region. The substructures of the two infrared bands are analyzed in terms of different levels of connectivity of the water molecules, along the statements of the percolation model. Both band profiles appear to be related to the different degrees of connectivity of water molecules. Comparison of the data with the predictions of the percolation model shows good agreement as for the temperature evolution of liquid water. This work provides additional support to the interpretation of water bands substructures as signatures of its very specific connectivity pattern.

Measurement of coherent Debye-Waller factor in in vivo deuterated C-phycocyanin by inelastic neutron scattering.

Quasielastic neutron scattering measurements of dry and 35% D2O hydrated amorphous protein powder of C-phycocyanin were made as a function of temperature ranging from 313K down to 100K. The protein is grown from blue-green algae cultured in D2O and is deuterated up to 99%. The scattering is thus dominated by coherent scattering. Within the best energy resolution of the time-of-flight instrument, which is 28 mueV FWHM, the scattering appears entirely elastic. For this reason we are able to extract a coherent Debye-Waller factor by making an independent measurement of the static structure factor. We observe a considerable difference in the q dependence of the Debye-Waller factor between the dry and hydrated proteins; furthermore, there is an interesting temperature dependence of the Debye-Waller factor that is quite different from that predicted for dense hard-sphere liquids.

Dynamics of hydrogen atoms in superoxide dismutase by quasielastic neutron scattering.

The low energy dynamic of the enzyme Cu,Zn superoxide dismutase have been investigated by means of quasielastic neutron scattering in the temperature range 4-320 K. Below 200 K the scattering is purely elastic, while above this temperature a pronounced decrease in the elastic intensity is observed, together with the onset of a small quasielastic component. This behavior is similar to that previously observed in other more flexible globular proteins, and can be attributed to transitions between slightly different conformational substates of the protein tertiary structure. The presence of only a small quasielastic component, whose intensity is < or = 25% of the total spectrum, is related to the high structural rigidity of this protein.