Introduction

An amyloid fibril is a highly ordered, insoluble form of protein that results when a normally soluble protein aggregates via a self-association process to form structured nanotubes. Such fibrils can now be routinely generated from purified proteins in the laboratory, and have attracted interest due to their role in a variety of disease states. Their formation can be promoted under certain environmental conditions (typically those that lie outside the realms of physiological control) and this inherent ability to form ordered structures of nanoscale dimensions facilitates their development as novel biomaterials. Our research focuses on this new alternative direction in amyloid research.

Amyloid structure

The overall dimensions of a typical fibril nanotube can range between ∼80-130Å in diameter and up to several micrometres in length. The block arrows in the model, shown in figure 1 (based on the model by Jimenez et al, 1999), represent sections of defined structure arranged in a cross-beta pattern.

Figure 1: Model of an amyloid protein fibril

The extensive bonding network gives the structure remarkable strength. The final conformation of amyloid nanotubes show strikingly similar morphologies, despite the diverse nature of the proteins known to form amyloid. It is also important to note that while this general structure does not change significantly from protein to protein, or in different conditions, subtle differences in the dimensions of these nanotubes have been observed. Understanding how to control these dimensions is a key aspect in this field of research.

Mechanism of formation

The simplest possible nucleation-polymerisation mechanism describes a period of time (lag phase) where no amyloid exists and partially unfolded intermediate precursors (often monomeric units) are able to interact and dissociate freely. After a series of unfavourable association steps, a critical concentration of “nucleating” molecules (oligomers) may be reached, whereafter amyloid growth occurs rapidly until a final equilibrium concentration is reached. This model has implications, in that growth may be seeded and that slight changes in physiological conditions may trigger rapid fibril formation, via a shift in the equilibrium that exists for peptide associations in favour of fibril formation. Only under extreme non-physiological conditions has the last step in the reaction been shown to be reversible.

Amyloid Protein as a Basis for Nanomaterials

Protein molecules are attractive for development as biomaterials because of three factors: they are of appropriate physical size, they possess inherent specificity towards other compounds, and they are able to be functionalised. Additionally, amyloidogenic protein is very well ordered and strong, and has a unique tertiary structure possessing a form of natural “scaffolding”. They are also capable of spontaneous self-assembly and can be manipulated both structurally and functionally.

Materials and methods

Protein extraction

Wheat flour proteins were extracted using a modified Osborne fractionation, as previously described (Bushuk et al 1997). The protein extracts were dialysed against distilled water then freeze dried for use.

Amyloid fibril formation

Protein extracts were placed under various conditions, mostly low pH, known to favour amyloid formation (Meehan et al, 2004)

Results and discussion

Detection of amyloid protein

The presence of amyloid was determined using a variety of techniques, including physical appearance, dye-binding assay and X-ray diffraction.

Physical appearance

Fibrillar amyloid material possesses a characteristic morphology, which can be analysed using imaging techniques such as transmission electron microscopy (TEM). Figure 2 illustrates the structural similarities of amyloid fibrils formed from bovine insulin and SDS-insoluble glutenin.

Figure 2. Representative images obtained for bovine insulin incubated in 0.025 M HCl, 0.1 M NaCl at 50°C (left, scale bar = 100nm) and b) SDS-insoluble glutenin protein incubated in phosphate buffer (0.05M, 0.5% SDS (w/v)) at 25°C for up to 105 days (right, scale bar = 200 nm).

Dye-binding assay

Dye binding assays, such as the Thioflavin-T (ThT) assay, are considered indicative of the presence of amyloid protein. Figure 3 illustrates the dye-binding curves for insulin and glutenin protein.

Figure 3. Bovine insulin fluorescence emission intensity at 482 nm (arbitrary units, λ _Ex 450 nm), 30 μM ThT (top), glutenin protein fluorescence emission intensity at 482 nm (arbitrary units, bottom)

X-ray diffraction

X-ray fibre diffraction can establish if the “cross-beta” pattern indicative of amyloid is present in a protein sample. Figure 4 illustrates the x-ray fibre diffraction pattern for SDS-insoluble glutenin fibrils.

Figure 4: X-ray diffraction pattern of wheat glutenin

The axial diffraction band observed in the X-ray data indicates the presence of inter beta-strands (with a spacing of approx 4.7Å). The absence of a 10Å meridonal band should not be interpreted as negative for amyloid structure, as the absence of this band has been noted before (Sunde et al, 1997), particularly for proteins high in glutamine, such as glutenin.

Future directions for research

Although the results shown here are only preliminary, were are currently working toward determining the exact identity and nature of the glutenin proteins that are fibrilising. We are also looking at whether there are specific peptide sequences in wheat proteins that contribute toward fibril formation. Similarly, we are studying the influence of side-chain derivatisation on the propensity of proteins to fibrilise and on the structure of the amyloid fibrils that form. Further along in the research, we hope to be able to determine whether the cereal protein based fibrils can be incorporated into biomaterials such as protein-based films.

References

Bushuk, W., Hay, R.L., Larsen, N.G., Sara, R.G., Simmons, L.D., and Sutton, K.H. (1997). Cereal Chemistry 74:389-395.

Jimenez, J.L., Guijarro, J.I., Orlova, E., Zurdo, J., Dobson, C.M., Sunde, M., and Saibil, H.R. (1999). EMBO 18:815-821

Meehan S., Berry Y., Luisi B., Dobson C.M., Carver J.A., and MacPhee C.E. (2004). Journal of Biological Chemistry 279:3413-3419.

Sunde, M., Serpell, L.C., Bartlam, M., Fraser, P.E., Pepys, M.B. and Blake, C.F. (1997). Journal of Molecular Biology 273:729-739.