Cait E. MacPhee

Characterization of the nanoscale properties of individual amyloid fibrils

We report the detailed mechanical characterization of individual amyloid fibrils by atomic force microscopy and spectroscopy. These self-assembling materials, formed here from the protein insulin, were shown to have a strength of 0.6 ± 0.4 GPa, comparable to that of steel (0.6–1.8 GPa), and a mechanical stiffness, as measured by Youngs modulus, of 3.3 ± 0.4 GPa, comparable to that of silk (1–10 GPa). The values of these parameters reveal that the fibrils possess properties that make these structures highly attractive for future technological applications. In addition, analysis of the solution-state growth kinetics indicated a breakage rate constant of 1.7 ± 1.3 × 10−8 s−1, which reveals that a fibril 10 μm in length breaks spontaneously on average every 47 min, suggesting that internal fracturing is likely to be of fundamental importance in the proliferation of amyloid fibrils and therefore for understanding the progression of their associated pathogenic disorders.

Atomic structure and hierarchical assembly of a cross-β amyloid fibril.

The cross-β amyloid form of peptides and proteins represents an archetypal and widely accessible structure consisting of ordered arrays of β-sheet filaments. These complex aggregates have remarkable chemical and physical properties, and the conversion of normally soluble functional forms of proteins into amyloid structures is linked to many debilitating human diseases, including several common forms of age-related dementia. Despite their importance, however, cross-β amyloid fibrils have proved to be recalcitrant to detailed structural analysis. By combining structural constraints from a series of experimental techniques spanning five orders of magnitude in length scale—including magic angle spinning nuclear magnetic resonance spectroscopy, X-ray fiber diffraction, cryoelectron microscopy, scanning transmission electron microscopy, and atomic force microscopy—we report the atomic-resolution (0.5 Å) structures of three amyloid polymorphs formed by an 11-residue peptide. These structures reveal the details of the packing interactions by which the constituent β-strands are assembled hierarchically into protofilaments, filaments, and mature fibrils.

Molecular conformation of a peptide fragment of transthyretin in an amyloid fibril

The molecular conformation of peptide fragment 105–115 of transthyretin, TTR(105–115), previously shown to form amyloid fibrils in vitro, has been determined by magic-angle spinning solid-state NMR spectroscopy. 13C and 15N linewidth measurements indicate that TTR(105–115) forms a highly ordered structure with each amino acid in a unique environment. 2D 13C-13C and 15N-13C-13C chemical shift correlation experiments, performed on three fibril samples uniformly 13C,15N-labeled in consecutive stretches of 4 aa, allowed the complete sequence-specific backbone and side-chain 13C and 15N resonance assignments to be obtained for residues 105–114. Analysis of the 15N, 13CO, 13Cα, and 13Cβ chemical shifts allowed quantitative predictions to be made for the backbone torsion angles φ and ψ. Furthermore, four backbone 13C–15N distances were determined in two selectively 13C,15N-labeled fibril samples by using rotational-echo double-resonance NMR. The results show that TTR(105–115) adopts an extended β-strand conformation that is similar to that found in the native protein except for substantial differences in the vicinity of the proline residue.

Modelling amyloid fibril formation kinetics: mechanisms of nucleation and growth

Amyloid and amyloid-like fibrils are self-assembling protein nanostructures, of interest for their robust material properties and inherent biological compatibility as well as their putative role in a number of debilitating mammalian disorders. Understanding fibril formation is essential to the development of strategies to control, manipulate or prevent fibril growth. As such, this area of research has attracted significant attention over the last half century. This review describes a number of different models that have been formulated to describe the kinetics of fibril assembly. We describe the macroscopic implications of mechanisms in which secondary processes such as secondary nucleation, fragmentation or branching dominate the assembly pathway, compared to mechanisms dominated by the influence of primary nucleation. We further describe how experimental data can be analysed with respect to the predictions of kinetic models.

Mimicking phosphorylation of αB-crystallin affects its chaperone activity

AlphaB-crystallin is a member of the sHsp (small heat-shock protein) family that prevents misfolded target proteins from aggregating and precipitating. Phosphorylation at three serine residues (Ser19, Ser45 and Ser59) is a major post-translational modification that occurs to alphaB-crystallin. In the present study, we produced recombinant proteins designed to mimic phosphorylation of alphaB-crystallin by incorporating a negative charge at these sites. We employed these mimics to undertake a mechanistic and structural investigation of the effect of phosphorylation on the chaperone activity of alphaB-crystallin to protect against two types of protein misfolding, i.e. amorphous aggregation and amyloid fibril assembly. We show that mimicking phosphorylation of alphaB-crystallin results in more efficient chaperone activity against both heat-induced and reduction-induced amorphous aggregation of target proteins. Mimick-ing phosphorylation increased the chaperone activity of alphaB-crystallin against one amyloid-forming target protein (kappa-casein), but decreased it against another (ccbeta-Trp peptide). We observed that both target protein identity and solution (buffer) conditions are critical factors in determining the relative chaperone ability of wild-type and phosphorylated alphaB-crystallins. The present study provides evidence for the regulation of the chaperone activity of alphaB-crystallin by phosphorylation and indicates that this may play an important role in alleviating the pathogenic effects associated with protein conformational diseases.

Giving structure to the biofilm matrix: an overview of individual strategies and emerging common themes

Biofilms are communities of microbial cells that underpin diverse processes including sewage bioremediation, plant growth promotion, chronic infections and industrial biofouling. The cells resident in the biofilm are encased within a self-produced exopolymeric matrix that commonly comprises lipids, proteins that frequently exhibit amyloid-like properties, eDNA and exopolysaccharides. This matrix fulfils a variety of functions for the community, from providing structural rigidity and protection from the external environment to controlling gene regulation and nutrient adsorption. Critical to the development of novel strategies to control biofilm infections, or the capability to capitalize on the power of biofilm formation for industrial and biotechnological uses, is an in-depth knowledge of the biofilm matrix. This is with respect to the structure of the individual components, the nature of the interactions between the molecules and the three-dimensional spatial organization. We highlight recent advances in the understanding of the structural and functional role that carbohydrates and proteins play within the biofilm matrix to provide three-dimensional architectural integrity and functionality to the biofilm community. We highlight, where relevant, experimental techniques that are allowing the boundaries of our understanding of the biofilm matrix to be extended using Escherichia coli, Staphylococcus aureus, Vibrio cholerae, and Bacillus subtilis as exemplars.

Altered aggregation properties of mutant γ-crystallins cause inherited cataract

Protein inclusions are associated with a diverse group of human diseases ranging from localized neurological disorders through to systemic non‐neuropathic diseases. Here, we present evidence that the formation of intranuclear inclusions is a key event in cataract formation involving altered γ‐crystallins that are un likely to adopt their native fold. In three different inherited murine cataracts involving this type of γ‐crystallin mutation, large inclusions containing the altered γ‐crystallins were found in the nuclei of the primary lens fibre cells. Their formation preceded not only the first gross morphological changes in the lens, but also the first signs of cataract. The inclusions contained filamentous material that could be stained with the amyloid‐detecting dye, Congo red. In vitro, recombinant mutant γB‐crystallin readily formed amyloid fibrils under physiological buffer conditions, unlike wild‐type protein. These data suggest that this type of cataract is caused by a mechanism involving the nuclear targeting and deposition of amyloid‐like inclusions. The mutant γ‐crystallins initially disrupt nuclear function, but then this progresses to a full cataract phenotype.

Functionalised amyloid fibrils for roles in cell adhesion

We describe experiments designed to explore the possibility of using amyloid fibrils as new nanoscale biomaterials for promoting and exploiting cell adhesion, migration and differentiation in vitro. We created peptides that add the biological cell adhesion sequence (RGD) or a control sequence (RAD) to the C-terminus of an 11-residue peptide corresponding to residues 105-115 of the amyloidogenic protein transthyretin. These peptides readily self-assemble in aqueous solution to form amyloid fibrils, and X-ray fibre diffraction shows that they possess the same strand and sheet spacing in the characteristic cross-beta structure as do fibrils formed by the parent peptide. We report that the fibrils containing the RGD sequence are bioactive and that these fibrils interact specifically with cells via the RGD group displayed on the fibril surface. As the design of such functionalized fibrils can be systematically altered, these findings suggest that it will be possible to generate nanomaterials based on amyloid fibrils that are tailored to promote interactions with a wide variety of cell types.

BslA is a self-assembling bacterial hydrophobin that coats the Bacillus subtilis biofilm

Biofilms represent the predominant mode of microbial growth in the natural environment. Bacillus subtilis is a ubiquitous Gram-positive soil bacterium that functions as an effective plant growth-promoting agent. The biofilm matrix is composed of an exopolysaccharide and an amyloid fiber-forming protein, TasA, and assembles with the aid of a small secreted protein, BslA. Here we show that natively synthesized and secreted BslA forms surface layers around the biofilm. Biophysical analysis demonstrates that BslA can self-assemble at interfaces, forming an elastic film. Molecular function is revealed from analysis of the crystal structure of BslA, which consists of an Ig-type fold with the addition of an unusual, extremely hydrophobic “cap” region. A combination of in vivo biofilm formation and in vitro biophysical analysis demonstrates that the central hydrophobic residues of the cap are essential to allow a hydrophobic, nonwetting biofilm to form as they control the surface activity of the BslA protein. The hydrophobic cap exhibits physiochemical properties remarkably similar to the hydrophobic surface found in fungal hydrophobins; thus, BslA is a structurally defined bacterial hydrophobin. We suggest that biofilms formed by other species of bacteria may have evolved similar mechanisms to provide protection to the resident bacterial community.

Protein folding and misfolding: a paradigm of self-assembly and regulation in complex biological systems.

Understanding biological complexity is one of the grand scientific challenges for the future. A living organism is a highly evolved system made up of a large number of interwoven molecular networks. These networks primarily involve proteins, the macromolecules that enable and control virtually every chemical process that takes place in the cell. Proteins are also key elements in the essential characteristic of living systems, their ability to function and replicate themselves through controlled molecular interactions. Recent progress in understanding the most fundamental aspect of polypeptide self–organization, the process by which proteins fold to attain their active conformations, provides a global platform to gain knowledge about the function of biological systems and the regulatory mechanisms that underpin their ability to adapt to changing conditions. In order to exploit such progress effectively, we are developing a variety of approaches, including procedures that use experimental data to restrain the properties of complex systems in computer simulations, to describe their behaviour under a wide variety of conditions. We believe that such approaches can lead to significant advances in understanding biological complexity, in general, and protein folding and misfolding in particular. These advances would contribute to: a more effective exploitation of the information from genome sequences; more rational therapeutic approaches to diseases, particularly those associated with ageing; the responsible control of our own evolution; and the development of new technologies based on mimicking the principles of biological self–assembly, for instance in nanotechnology. More fundamentally, we believe that this research will result in a more coherent understanding of the origin, evolution and functional properties of living systems.