Amyloid fibril forming proteins have been related to some neurodegenerative diseases and are not fully understood. In some such systems, these amyloid fibrils have been found to form radially oriented spherulite structures. The thermal dependence of formation and growth of these spherulite structures in two model protein systems, beta-lactoglobulin and insulin at low pH aqueous and high temperature conditions, have been monitored with time-lapse optical microscopy and quantified. A population-based polymerization reaction model was developed and applied to the experimental data with excellent agreement. While spherulites in the insulin solutions formed and grew at approximately 25x the rate of spherulites in the beta-lactoglobulin solutions, the temperature dependence and activation energies of both systems were found to be very similar to one another, suggesting that the underlying rate-limiting mechanisms for both formation and growth are consistent across the two systems. The similarity of both of these amyloid fibril forming protein systems provides confidence in their use as model systems for extrapolating understanding to similar systems involved in neurodegenerative diseases.

The aggregation of misfolded proteins into amyloid fibrils, and the importance of this step for various diseases, is well known. However, it is becoming apparent that the fibril is not the only structure that aggregating proteins of widely different types may adopt. Around the isoelectric point, when the net charge is essentially zero, rather monodisperse and quasi-amorphous nanoscale particles form. These particles are found to contain limited runs of beta-sheet structure, but their overall organization is random. These nanoparticles have the potential to be useful for such applications as the slow release of drugs. The amyloid fibrils form away from the isoelectric point, but over certain ranges of, e.g., pH, the fibrils themselves do not exist freely, but form suprafibrillar aggregates termed spherulites. These consist of fibrils radiating from a central nucleus, and form by new species attaching to the ends of growing fibrils, rather than by the aggregation of pre-existing fibrils. Under the polarizing light microscope, they exhibit a Maltese cross shape due to their symmetry. The rate of aggregation is determined by factors involving (at least) protein size, concentration, presence of salt and charge. The occurrence of spherulites, which have been found in vivo as well as in vitro, appears to be generic, although the factors which determine the equilibrium between free fibril and spherulite are not as yet clear.

The importance of misfolding proteins forming amyloid fibrils for the aetiology of many diseases, particularly those of old age, is well recognized. This phenomenon is now thought to be a universal property of proteins, as long as appropriate conditions for loosening the native folded structure can be found, which may be outside those of normal physiology. However, the beta-sheet-rich structure of the amyloid fibril does not need to exist in isolation. Recent work has shown that higher order assemblies of the fibrils occur into structures resembling spherulites found in common synthetic semi-crystalline polymers. In these, the fibrils grow outwards from an inner core, thought to be amorphous. Data will be presented on the kinetics of growth of these fibrils for different proteins, so that similarities and differences can be revealed, and related to subtle differences in appearance under the microscope. The in vitro assembly of amyloid fibrils is usually thought to occur well away from the isoelectric point of the protein, and these are the conditions under which they have most been studied. Around the isoelectric point, particulate self-assembly is known to occur for beta-lactoglobulin, and we can now show this is also a generic form of protein self-assembly once the net charge on the protein is close to zero. Nevertheless, the charge is not actually zero, and salt in the solution is found to have a significant effect on the growth of the particles. The use of SAXS, thioflavin T staining and FTIR also shows that within the particles there is also clear evidence for amyloid-like beta-sheet structure, particularly in the case when salt is absent, demonstrating that this particular motif underlies this very different form of protein self-assembly.

No abstract

When beta-lactoglobulin in low p H aqueous solutions is exposed to high temperature for extended time, spherulites composed of amyloid fibrils of the beta-lactoglobulin protein form. Many of these spherulites have fibrils that radiate out from a centre and, under crossed polarisers, exhibit a symmetric Maltese Cross structure. However, a significant fraction (50 of the 101 observed spherulites) of beta-lactoglobulin spherulites formed under these conditions demonstrate various forms of irregularity in apparent structure. The irregularities of spherulites structures were qualitatively investigated by comparing optical microscopy images observed under crossed polarisers to computationally produced images of various internal structures. In this way, inner spherulite structures are inferred from microscopy images. Modelled structures that were found to produce computed images similar to some of the experimentally viewed images include fibrils curving as they radiate from a single nucleation point; multiple spherulites nucleating in close proximity to one another; and fibrils curving in opposite directions above and below a single nucleation point.