Sally L. Gras

Metastability of native proteins and the phenomenon of amyloid formation.

An experimental determination of the thermodynamic stabilities of a series of amyloid fibrils reveals that this structural form is likely to be the most stable one that protein molecules can adopt even under physiological conditions. This result challenges the conventional assumption that functional forms of proteins correspond to the global minima in their free energy surfaces and suggests that living systems are conformationally as well as chemically metastable.

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.

Folic Acid Conjugated Amino Acid-Based Star Polymers for Active Targeting of Cancer Cells

Amino acid-based core cross-linked star (CCS) polymers (poly(L-lysine)(arm)poly(L-cystine)(core)) with peripheral allyl functionalities were synthesized by sequential ring-opening polymerization (ROP) of amino acid N-carboxyanhydrides (NCAs) via the arm-first approach, using N-(trimethylsilyl)allylamine as the initiator. Subsequent functionalization with a poly(ethylene glycol) (PEG)-folic acid conjugate via thiol-ene click chemistry afforded poly(PEG-b-L-lysine)(arm)poly(L-cystine)(core) stars with outer PEG coronas decorated with folic acid targeting moieties. Similarly, a control was prepared without folic acid, using just PEG. A fluorophore was used to track both star polymers incubated with breast cancer cells (MDA-MB-231) in vitro. Confocal microscopy and flow cytometry revealed that the stars could be internalized into the cells, and higher cell internalization was observed when folic acid moieties were present. Cytotoxicity studies indicate that both stars are nontoxic to MDA-MB-231 cells at concentrations of up to 50 μg/mL. These results make this amino acid-based star polymer an attractive candidate in targeted drug delivery applications including chemotherapy.

Dopamine and the Dopamine Oxidation Product 5,6-Dihydroxylindole Promote Distinct On-Pathway and Off-Pathway Aggregation of α-Synuclein in a pH-Dependent Manner

The deposition of alpha-synuclein (alpha-syn) aggregates in dopaminergic neurons is a key feature of Parkinsons disease. While dopamine (DA) can modulate alpha-syn aggregation, it is unclear which other factors can regulate the actions of DA on alpha-syn. In this study, we investigated the effect of solution conditions (buffer, salt and pH) on the oligomerization of alpha-syn by DA. We show that alpha-syn oligomerization is dependent on the oxidation of DA into reactive intermediates. Under acidic pH conditions, DA is stable, and DA-mediated oligomerization of alpha-syn is inhibited. From pH 7.0 to pH 11.0, DA is unstable and undergoes redox reactions, promoting the formation of SDS-resistant soluble oligomers of alpha-syn. We show that the reactive intermediate 5,6-dihydroxylindole mediates the formation of alpha-syn soluble oligomers under physiological conditions (pH 7.4). In contrast, under acidic conditions (pH 4.0), 5,6-dihydroxylindole promotes the formation of SDS-resistant insoluble oligomers that further associate to form sheet-like fibrils with beta-sheet structure that do not bind the dye thioflavin T. These results suggest that distinct reactive intermediates of DA, and not DA itself, interact with alpha-syn to generate the alpha-syn aggregates implicated in Parkinsons disease.

The effect of milk processing on the microstructure of the milk fat globule and rennet induced gel observed using confocal laser scanning microscopy.

Confocal laser scanning microscopy (CLSM) was successfully used to observe the effect of milk processing on the size and the morphology of the milk fat globule in raw milk, raw ultrafiltered milk, and standardized and pasteurized milk prepared for cheese manufacture (cheese-milk) and commercial pasteurized and homogenized milk. Fat globule size distributions for the milk preparations were analyzed using both image analysis and light scattering and both measurements produced similar data trends. Changes to the native milk fat globule membrane (MFGM) were tracked using a MFGM specific fluorescent stain that allowed MFGM proteins and adsorbed proteins to be differentiated on the fat globule surface. Sodium dodecyl sulfate polyacrylamide gel electrophoresis confirmed the identity of native MFGM proteins isolated from the surface of fat globules within raw, UF retentate, and cheese-milk preparations, whereas only casein was detected on the surface of fat globules in homogenized milk. The microstructure, porosity, and gel strength of the rennet induced gel made from raw milk and cheese-milk was also found to be comparable and significantly different to that made from homogenized milk. Our results highlight the potential use of CLSM as a tool to observe the structural details of the fat globule and associated membrane close to its native environment.

The Assembly of Individual Chaplin Peptides from Streptomyces coelicolor into Functional Amyloid Fibrils

The self-association of proteins into amyloid fibrils offers an alternative to the natively folded state of many polypeptides. Although commonly associated with disease, amyloid fibrils represent the natural functional state of some proteins, such as the chaplins from the soil-dwelling bacterium Streptomyces coelicolor, which coat the aerial mycelium and spores rendering them hydrophobic. We have undertaken a biophysical characterisation of the five short chaplin peptides ChpD-H to probe the mechanism by which these peptides self-assemble in solution to form fibrils. Each of the five chaplin peptides produced synthetically or isolated from the cell wall is individually surface-active and capable of forming fibrils under a range of solution conditions in vitro. These fibrils contain a highly similar cross-β core structure and a secondary structure that resembles fibrils formed in vivo on the spore and mycelium surface. They can also restore the growth of aerial hyphae to a chaplin mutant strain. We show that cysteine residues are not required for fibril formation in vitro and propose a role for the cysteine residues conserved in four of the five short chaplin peptides.

Multilayered microspheres for the controlled release of growth factors in tissue engineering.

Tissue regeneration may be stimulated by growth factors but to be effective, this delivery must be sustained and requires delivery vehicles that overcome the short half-life of these molecules in vivo. One promising approach is to couple growth factors to the biomaterial surface so that they are readily bioavailable. Here the layer-by-layer process was used to construct a multilayered polyelectrolyte delivery system on the surface of poly(lactic-co-glycolic) acid constructs. The system was first optimized on a planar surface before translation to a 3D microsphere system. The layers incorporated heparin to facilitate the loading of basic fibroblast growth factor and increase growth factor stability. Cross-linked capping layers also reduced any burst release. The model growth factor was released in a sustained manner and stimulated significantly higher cell proliferation in vitro on release compared with the addition of the growth factor heparin complex free in solution, demonstrating the promise of this approach.

Facile Pretreatment of Bacillus circulans β-Galactosidase Increases the Yield of Galactosyl Oligosaccharides in Milk and Lactose Reaction Systems

The commercially available preparation of beta-galactosidase from Bacillus circulans , known as Biolacta FN5, has been extensively used in the production of prebiotic galactooligosaccharides (GOS). This study focuses on characterizing the production of GOS in two reaction systems: 10% lactose (w/v) in buffer and skim milk. Analysis of the temperature dependence of the GOS yield along with the relative rates of GOS synthesis and degradation leads to the finding that GOS degradation activity was selectively decreased in Biolacta FN5 above 40 degrees C. Facile heat treatment of Biolacta FN5 solution prior to use allowed for GOS yields to be significantly increased in both reaction systems.

Effect of the Substrate Concentration and Water Activity on the Yield and Rate of the Transfer Reaction of β-Galactosidase from Bacillus circulans

Prebiotic galactosyl oligosaccharides (GOS) are produced from lactose by the enzyme β-galactosidase. It is widely reported that the highest GOS levels are achieved when the initial lactose concentration is as high as possible; however, little evidence has been presented to explain this phenomenon. Using a system composed of the commercial β-galactosidase derived from Bacillus circulans known as Biolacta FN5, lactose and sucrose, the relative contribution of water activity, and substrate availability were assessed. Oligosaccharide levels did not appear to be affected by changes in water activity between 1.0 and 0.77 at a constant lactose concentration. The maximum oligosaccharide concentration increased at higher initial concentrations of lactose and sucrose, while initial reaction rates for transfer increased but remained constant for hydrolysis. This suggests that the high oligosaccharide levels achieved at the raised initial saccharide concentration are due to increases in reactions that form oligosaccharides rather than decreases in concurrent reactions, which degrade oligosaccharides. There were different effects from changing the initial concentration of lactose compared to sucrose, suggesting that the ability of lactose to act as a donor saccharide may be more important for increasing maximum oligosaccharide concentrations than the combined ability of both saccharides to act as galactosyl acceptors.

Amyloid fibrils nucleated and organized by DNA origami constructions

Amyloid fibrils are ordered, insoluble protein aggregates that are associated with neurodegenerative conditions such as Alzheimer’s disease1. The fibrils have a common rod-like core structure, formed from an elongated stack of β-strands, and have a rigidity similar to silk (Young’s modulus of 0.2-14 Gpa)2. They also exhibit high thermal and chemical stability3, and can be assembled in vitro from short synthetic non-disease-related peptides4,5. As a result, they are of significant interest in the development of self-assembled materials for bionanotechnology applications6. Synthetic DNA molecules have previously been used to form intricate structures and organize other materials such as metal nanoparticles7,8, and could in principle be used to nucleate and organize amyloid fibrils. Here we show that DNA origami nanotubes can sheathe amyloid fibrils formed within them. The fibrils are built by modifying the synthetic peptide fragment corresponding to residues 105-115 of the amyloidogenic protein transthyretin (TTR)9, and a DNA origami10 construct is used to form 20-helix DNA nanotubes with sufficient space for the fibrils inside. Once formed, the fibril-filled nanotubes can be organized onto predefined two-dimensional platforms via DNA-DNA hybridization interactions.