Newsletter - Edition 27

Protein Misfolding and Aggregation: From Diseases to Devices

Oxford Innovation Society Seminar - January 1999
Chaired by Professor Chris Dobson, Director of the Oxford Centre for Molecular Sciences

In order to function, a protein must fold to a specific conformation following synthesis on the ribosome. This folding process is therefore the second stage of the translation of genetic information into biological activity. For many years this phenomenon was considered a scientific curiosity, but is now recognised as central to an understanding of many aspects of molecular and cellular biology, including molecular trafficking and the control of the cell cycle. In addition, the failure of proteins to fold correctly, or the subsequent misfolding of correctly folded proteins, is increasingly associated with diseases ranging from cystic fibrosis to Alzheimer's and CJD. This Seminar day will describe our present knowledge of the mechanisms of protein folding, based particularly on the results of NMR experiments coupled with other biophysical measurements and with theoretical simulations. In addition it will address the question of protein misfolding and the possibility of novel therapeutic strategies to prevent or combat the diseases associated with this phenomenon.

The Properties of Denatured Proteins

Dr Lorna Smith - Random Coils and Unfolded Proteins

Non-native states of proteins are of increasing interest because of their relevance to issues such as protein folding, stability translocation and aggregation. However, defining the conformational properties of denatured and partly folded proteins is challenging as these states are in general an ensemble of interconverting conformers. One approach that we have been developing to give insight into these systems uses a combination of experimental procedures and theoretical models. My presentation will concentrate on this strategy and will show how, by applying high resolution heteronuclear nuclear magnetic resonance techniques to non-native proteins, a wealth of experimental data can be collected under favourable conditions. The interpretation of these data by comparison with predictions from theoretical models will be presented, considering a statistical model for a random coil and results from molecular dynamics simulations.

The statistical model for a random coil uses the distribution of torsion angles in a database of native folded protein structures to provide a description of the local conformational preferences of each residue in a random coil. Good agreement has been found between predictions from this model and experimental NMR parameters, including coupling constants, NOE intensities and chemical shift differences. I will illustrate the approach with experimental NMR data for short peptides, lysozyme denatured in 8M urea and fibronectin binding protein. This latter protein is of particular interest as it is unfolded but biologically active under physiological conditions.

Molecular dynamics simulations provide a method for generating models for the more global properties of non-native protein conformations. I will demonstrate this with results from some MD simulations of peptide fragments from the hen lysozyme sequence, comparing the predictions with data from experimental NMR studies of these peptides. These simulations give insight into the behaviour of polypeptide chains under strongly denaturing conditions. Of particular interest from this work is the observation of residual native-like secondary structure in the C-terminal part of the lysozyme sequence being stabilised by non-native interactions.

Dr Christina Redfield - Compact States and Molten Globules

A major goal in attempts to understand the events occurring during protein folding is to define the structures of species intermediate between the fully folded and fully unfolded states. These partly-folded states range from proteins which resemble native states in both their secondary structure and tertiary interactions to those which have very few native-like interactions and resemble unfolded proteins. In between these two extremes lies the classic molten globule, a partly-folded species with native-like secondary structure but lacking persistent tertiary interactions. The focus of our research is to understand, at the molecular level, the nature and stability of partly-folded proteins.

Nuclear magnetic resonance (NMR) spectroscopy is a powerful technique for the study of the structure and dynamics of fully folded proteins in solution. Partly-folded proteins, such as molten globules, have been difficult to study using high resolution NMR techniques because of the conformational heterogeneity and lack of specific tertiary contacts of these states. These effects can give rise to poor chemical shift dispersion in the NMR spectrum, to averaging of NMR parameters, and to extreme line broadening of NMR resonances which can preclude the application of state-of-the-art 2D and 3D NMR techniques.

alpha-Lactalbumin (a-LA) is a 123-residue two-domain calcium-binding protein that behaves as a classic molten globule under a variety of conditions. The 2-D HSQC NMR spectrum of the a-LA molten globule at pH 2 contains only 3 peaks which arise from the N-terminal residues; the remaining resonances are too broad to be observed. The molten globule can be unfolded in a stepwise manner by the addition of urea; this unfolding is accompanied by a dramatic increase in the number of resolved peaks. The sharp resonances observed at each urea concentration correspond to the regions of the structure that are unfolded. There is a strong correlation between the native structure and the pattern of unfolding of the molten globule in urea. Signals corresponding to the two domains of the protein in the native state become resolved in the spectrum of the partly-folded protein at different urea concentrations. In addition to the difference in unfolding pattern between the two domains, there are differences between the urea concentrations at which the individual helices appear to unfold.

The alpha-lactalbumin molten globule unfolds in a non-cooperative manner in urea. The power of the NMR method is that it can probe unfolding at the level of individual residues. These studies are providing insight into the nature and stability of the compact molten globule states of proteins.

Protein Aggregation and Disease

Dr Margaret Sunde - Amyloid Formation and Disease

A number of human diseases have recently been shown to be due to incorrect protein folding. The amyloid diseases are disorders of protein folding in which proteins and peptides take up an incorrect, fibrillar structure. This group of disorders includes Alzheimers's disease, bovine spongiform encephalopathy (BSE) and type II diabetes. The molecular structure of amyloid has been investigated by X-ray fibre diffraction studies and electron microscopy. All amyloid fibrils, regardless of the disease and protein involved, have a similar morphology and beta-sheet core structure.

Studies of amyloid fibril formation involving amyloidogenic lysozyme and transthyretin variants were described. Lysozyme has extensive helical native structure, yet this protein forms fibrils very similar to those composed of transthyretin which is a beta-sheet protein. The common features shared by amyloid-forming variants of these proteins are a destabilisation of the native protein fold, enhanced conformational dynamics and a reduction in the extent of stable secondary structure. These characteristics result in the population of partially folded, aggregation-prone forms of the proteins, which can associate intermolecularly, in an ordered fashion, to produce amyloid fibrils, as illustrated schematically in the figure above.

Dr Jesús Zurdo - Structure and Properties of SH3 Amyloid Fibrils

The SH3 domain of the bovine phosphatidylinositol 3'-kinase (PI3-SH3) forms amyloid fibrils when incubated at low pH for long periods of time. These fibrils show similar characteristics to those exhibited by well known pathological amyloids, this makes the SH3 domain a good model to study the processes that undergo fibril formation, as well as the structure and properties of amyloid fibrils.

SH3 fibrils show a number of different morphologies depending several factors, like pH, salts, buffer and time. On the other hand, fibril formation can be accelerated if some pre-existing fibrils are present ('seeding'). Seeded fibrils adopt an overall straight morphology that contrast with a more flexible one exhibited by non-seeded preparations. FTIR studies suggest secondary structure variations between different fibril preparations. Relationships between fibril morphology and FTIR features are currently under analysis.

The three-dimensional structure of an amyloid fibril formed by the PI3-SH3 has been determined at 25 Å resolution using cryo-electron microscopy. The structure of the fibril is a double helix of two protofilament pairs wound around a hollow core. It shows a crossover repeat of ~600 Å and an axial subunit repeat of ~27 Å. The domain must unfold to adopt a longer, thinner shape to fit the amyloid form. The dimensions of the protofilaments (20 x 40 Å) can only accommodate a pair of flat b-sheets stacked against each other, with very little inter-strand twist. More studies are being carried out to get insight into b-strand arrangement and protofilament connections. This structure constitutes a good model to better understand the assembly of other amyloid fibrils.

New Directions and New Opportunities

Dr Fabrizio Chiti - Producing Ordered Aggregates to Order

We have been able to convert a small alpha/beta protein, acylphosphatase, from its soluble and native form into insoluble amyloid fibrils of the type observed in a range of pathological conditions. This was achieved by allowing slow growth in a solution containing moderate concentrations of trifluoroethanol. When analysed with electron microscopy the protein aggregate present in the sample after long incubation times consisted of extended, unbranched filaments of 30-50 Å in width that assemble into higher-order structures. This fibrillar material possesses extensive beta-sheet structure as revealed by far-UV circular dichroism and infra-red spectroscopy. Furthermore, the fibrils exhibit Congo red birefringence, increased fluorescence with thioflavin T and cause a red-shift of the Congo Red absorption spectrum.

All these characteristics are typical of amyloid fibrils. The results indicate that formation of amyloid occurs when the native fold of a protein is destabilised under conditions where non-covalent interactions, and in particular hydrogen bonding, within the polypeptide chain remain favourable. Preliminary studies aimed at elucidating the mechanisms of fibril formation suggest that the fibrillogenesis process of acylphosphatase is similar to that of the alpha/beta peptides and other proteins that are known to be associated with amyloidotic diseases. We suggest therefore that amyloid formation is not restricted to a small number of protein sequences but is a property common to many, if not all, polypeptide chains under appropriate conditions.

Dr Carol Robinson - Protein-protein Interactions and the Mechanism of Amyloid Fibril Formation

A key question in amyloid fibril formation is the nature of the protein-protein interactions and the structural transitions that occur during the build up of amyloid fibrils. Using mass spectrometry we have developed methods to enable us to probe protein-protein interactions by careful transfer from solution to gas phase.

Under critical mass spectrometry conditions we have observed the native hexameric and tetrameric states of insulin and transthyretin respectively. This has allowed us to look at the effects of pH, ionic strength and ligand binding on these complexes and to derive a relationship with their solution phase stabilities. In addition variation of the solution conditions has enabled us to probe the disassembly of these complexes to monomeric protein.

Structural changes within these monomeric species have been proposed to give rise to intermediates in the fibril formation process. Using hydrogen exchange labelling techniques we have looked at the structural conversion of monomeric protein to amyloidogenic intermediate. In addition, under solution conditions that promote fibril formation, we observe aggregates of these proteins in the gas phase. Taken together these results allow us to begin to explore the relationship between the size of aggregates and the kinetics of fibril formation as well as the structure of amyloidogenic intermediates.