Mechanisms of structure formation in particulate gels of beta-lactoglobulin formed near the isoelectric point.

Particulate gels are known to be formed by bovine beta-lactoglobulin near the isoelectric point when partial unfolding is allowed to occur under heating. The aggregation process of the protein has been investigated within the context of a nucleation and growth process by preparing gels under precisely controlled thermal histories. This was achieved using a Differential Scanning Calorimeter (DSC) to provide controlled heating rates, and known final temperatures and incubation times. The resulting particulate gels were characterized by their particle size and polydispersity using Environmental Scanning Electron Microscopy (ESEM), which permits hydrated samples to be observed. Particle size was found to decrease with increasing final temperature, with the aggregation taking longer to reach completion for lower temperatures. Particle size was also found to decrease with increasing heating rate. This system could be modelled as evolving via nucleation and growth by taking into account the fact that the concentration of the aggregating species was varying as a function of temperature as well as time. The intrinsic tryptophan fluorescence as a function of temperature was used as a guide to the fraction of unfolded protein in solution, thereby permitting successful comparisons between the model predictions and the particle sizes to be made.-1.

The binding of thioflavin-T to amyloid fibrils: localisation and implications.

Amyloid fibrils are a polymeric form of protein, involving a continuous beta-sheet with the strands perpendicular to the long axis of the fibril. Although typically implicated in diseases such as Alzheimer's disease and the transmissible spongiform encephalopathies, non disease-associated protein can also be converted into amyloid fibrils. Traditionally, amyloid fibrils are identified via the use of specific dyes such as Congo red and thioflavin-T, although their specificity is ill understood. Recently, solutions of bovine insulin and bovine beta-lactoglobulin have been found to form spherulites, micron-sized spherical structures containing radially arranged amyloid fibrils. When studied by confocal microscopy using polarised laser light and thioflavin-T, a consistent pattern of emission, rather than a uniform disc, was observed. This suggests the dye binds in a specific, regular fashion to amyloid fibrils. Confocal microscopy studies of thioflavin-T aligned in stretched poly-vinyl alcohol films showed that the dye dipole excitation axis lies parallel to the long molecular axis. Therefore, thioflavin-T binds to amyloid fibrils such that their long axes are parallel. We propose binding occurs in 'channels' that run along the length of the beta-sheet. Steric interactions between dye molecules and side chains indicate why thioflavin-T fluoresces more intensely when bound to amyloid fibrils and can explain why this interaction with amyloid fibrils is specific, but with varying efficiency.

The mechanism of amyloid spherulite formation by bovine insulin.

The formation of amyloid-containing spherulite-like structures has been observed in some instances of amyloid diseases, as well as in amyloid fibril-containing solutions in vitro. In this article we describe the structure and kinetics of bovine insulin amyloid fibril spherulites formed in the presence and absence of different salts and at different salt concentrations. The general spherulite structure consists of radially oriented amyloid fibrils, as shown by optical microscopy and environmental scanning electron microscopy. In the center of each spherulite, a "core" of less regularly oriented material is observed, whose size decreases when the spherulites are formed in the presence of increasing concentrations of NaCl. Similarly, amyloid fibrils form faster in the presence of NaCl than in its absence. A smaller enhancement of the rate of formation with salt concentration is observed for spherulites. These data suggest that both amyloid fibril formation and random aggregation occur concurrently under the conditions tested. Changes in their relative rates result in the different-sized cores observed in the spherulites. This mechanism can be likened to that leading to the formation of spherulites of polyethylene, in agreement with observations that polypeptide chains under partially denaturing conditions can exhibit behavior not dissimilar to that of synthetic polymers.

Formation and seeding of amyloid fibrils from wild-type hen lysozyme and a peptide fragment from the beta-domain.

Wild-type hen lysozyme has been converted from its soluble native state into highly organized amyloid fibrils. In order to achieve this conversion, conditions were chosen to promote partial unfolding of the native globular fold and included heating of low-pH solutions and addition of organic solvents. Two peptides derived from the beta-sheet region of hen lysozyme were also found to form fibrils very readily. The properties and morphologies of the amyloid fibrils formed by incubation either of the protein or the peptides are similar to those produced from the group of proteins associated with clinical amyloidoses. Fibril formation by hen lysozyme was substantially accelerated when aliquots of solutions in which fibrils of either one of the peptides or the full-length protein had previously formed were added to fresh solutions of the protein, revealing the importance of seeding in the kinetics of fibril formation. These findings support the proposition that the beta-domain is of particular significance in the formation of fibrils from the full-length protein and suggest similarities between the species giving rise to fibril formation and the intermediates formed during protein folding.