Physical chemistry of protein misfolding and amyloid formation


Project Leader: David Dunstan
Primary Contact: David Dunstan (davided@unimelb.edu.au)
Keywords: protein assembly
Disciplines: Chemical & Biomolecular Engineering
Domains: Convergence of engineering and IT with the life sciences

We propose to use recent advances in single molecule force measurement, metrology and spectroscopy to understand the following processes associated with protein misfolding:

  • The mechanism of amyloid formation.
  • The role of heavy metals, surfactants and hydrophobic molecules in amyloid formation.
  • The effect of amyloid fibres on membrane structure.

We also aim to develop novel methods for protein immobilisation and fluorescence imaging at interfaces.

The biological function of protein molecules is critically dependent on the three-dimensional arrangement of the constituent atoms. It is now known that several major human diseases are associated with abnormal protein conformations and associations which inhibit normal biochemical function (protein misfolding). Alzheimer’s disease, Parkinson’s disease, some forms of heart disease, type II diabetes and the infectious prion diseases such as Creutzfeldt-Jakob disease (CJD) are significant diseases that are associated with misfolded proteins1,2,3,4,5,6.

The three-dimensional arrangement of the constituent atoms of a protein molecule (the conformation) is largely controlled by relatively weak forces (hydrogen-bonds) so that proteins can, and do adopt a variety of different conformations. The probability of a protein molecule adopting a particular conformation depends not only on the amino acid sequence and the solution conditions, but also on the history of the protein. Energetic barriers between conformations make the transitions between certain conformations very unlikely. Thus a protein molecule, that at some time in its history has been led down the wrong folding path, may be trapped and unable to adopt a useful conformation. Furthermore, such a misfolded protein can act as a template or catalyst for the conversion of other protein molecules into a misfolded state.  In this sense, misfolded proteins can be infective disease agents, manifested, for example as CJD and mad cow disease 7.

In Alzheimer’s and Huntington’s disease, the misfolded protein forms insoluble fibrils, called amyloid fibrils, that are rich in antiparallel b-sheet structure, and are easily recognised due to their incorporation of dyes such as congo red and thioflavin T 8.  In fact, many proteins with disparate structures (including homopolymers4 and unnatural peptides) form amyloid fibrils.  This observation led Dobson to suggest that the antiparallel b-sheet conformation is driven by the peptide backbone rather than by a particular sequence of side-chains, and is the thermodynamically stable state of most proteins 5.  The corollary to this hypothesis is that the biologically active states of most proteins are only metastable. Therefore the study of the kinetics of protein folding, and the factors (chaperones, ions, surfactants etc) associated with protein misfolding will lead to an improved understanding of the amyloid and prion diseases.

The principle objective of this proposal is to use state-of-the-art methods in physical chemistry at the single molecule level namely: force measurement, metrology and spectroscopy to elucidate the mechanism of amyloid formation. Proteins are very complex molecules with many energy states. The kinetics of transformation from one state to another is dictated by the energy barrier between the two states.  We propose to measure the energy of transformation between the amyloid, native, and other states by using an Atomic Force Microscope to tease apart the protein. In this study we aim to measure the main features of this ‘energy landscape’ at the single molecule level.

(1)        Dobson, C.M. Nature 2003, 426, 884-990.

(2)        Sunde, M, Blake, C.C.F., Quarterly Reviews of Biophysics, 1998, 31, 1-39.

(3)        Collins, S. R., A. Douglass, et al. 2004. PLoS Biology 2(10): 1582-1590.

(4)        Bucciantini, M., Giannoni, E., Chiti, F., Baroni, F., Formigli, L., Zurdo, J., Taddei, N., Ramponi, G., Dobson, C.M. & Stefani, M. 2002 Nature 416, 507–511.

(5)        Dobson, C. M.1999.  Trends in Biochemical Sciences 24: 329-333.

(6)        Jahn.T.R. and Radford S. 2005. FEBs Journal 272: 5962-5970

(7)        Prusiner, S. 1998Proc. Natl. Acad. Sci. USA 95: 13363-13383.