Owen G. Jones

Inhibiting, promoting, and preserving stability of functional protein fibrils

Protein fibrils are relevant not only in medicine and amyloid-related neurodegenerative diseases, but also as functional structures in material science or biology. The assembly of protein into fibrils can be promoted or inhibited based on the chosen environmental conditions and interaction with suitable components. We review here the key strategies for promotion and inhibition of protein fibrillation in both physiological and non-physiological conditions in order to create functional designs. The major variables discussed are solvent conditions, metals/ions, biopolymers, aromatic compounds, and surface active components. Due to bias in research directions, deeper investigation has traditionally been carried out for inhibition of fibrillation, but focus has recently shifted. Thus, while various strategies are presented on the breakdown of mature protein fibrils, emphasis is given to the approaches leading to increased rigidity and length of resultant fibrils. We highlight important areas in this field that require further development and promising lines of future experiments.

Complexation of beta-Lactoglobulin Fibrils and Sulfated Polysaccharides

Fibrils of β-lactoglobulin, formed by heating at pH 2, were titrated with a sulfated polysaccharide (κ-carrageenan) to determine the morphology and mechanism of complex formation at low pH. Structural information on the resultant complexes was gathered using transmission electron microscopy, atomic force microscopy, Doppler electrophoresis, and small-angle neutron scattering. Electrophoresis demonstrated that the carrageenan complexed with protein fibrils until reaching a maximum complexation efficiency at a protein/polysaccharide (r) weight ratio of 5:3. Neutron scattering and microscopy indicated an increasing formation of spherical aggregates attached along the protein fibrils with increases in the carrageenan concentration. These globular particles had an average diameter of 30 nm. Small-angle neutron scattering of these complexes could be accurately described by a form factor corresponding to multistranded twisted ribbons with spherical aggregates along their contour length, arranged in a necklace configuration.

Fibrillation of β-Lactoglobulin at Low pH in the Presence of a Complexing Anionic Polysaccharide

The influence of electrostatic complexation with κ-carrageenan was tested on the fibrillation process of β-lactoglobulin at pH 2.0. Morphology and structural development were monitored through cross correlation dynamic light scattering, transmission electron microscopy, and atomic force microscopy. Scattering indicated that noncomplexed β-lactoglobulin monomers aggregated to form fibrils after 15-90 min of heating at 90 °C. However, electrostatic protein-carrageenan complexes found in the unheated system were unchanged by the thermal process. Images and scattering results showed that carrageenan complexes slowed fibrillation kinetics, possibly through reduction in available monomer concentration. Complexes adhered to fibrils at ends and junctions in TEM images, indicating interactive affinity with the fibers, presumably as heterogeneous nucleation sites.

Dynamic and viscoelastic interfacial behavior of β-lactoglobulin microgels of varying sizes at fluid interfaces

HYPOTHESIS Microgel particles formed from the whey protein β-lactoglobulin are able to stabilize emulsion and foam interfaces, yet their interfacial properties have not been fully characterized. Smaller microgels are expected to adsorb to and deform at the interface more rapidly, facilitating the development of highly elastic interfaces. METHODS Microgels were produced by thermal treatment under controlled pH conditions. Dynamic surface pressure and dilatational interfacial rheometry measurements were performed on heptane-water droplets to examine microgel interfacial adsorption and behavior. Langmuir compression and atomic force microscopy were used to examine the changes in microgel and monolayer characteristics during adsorption and equilibration. FINDINGS Microgel interfacial adsorption was influenced by bulk concentration and particle size, with smaller particles adsorbing faster. Microgel-stabilized interfaces were dominantly elastic, and elasticity increased more rapidly when smaller microgels were employed as stabilizers. Interfacial compression increased surface pressure but not elasticity, possibly due to mechanical disruption of inter-particle interactions. Monolayer images showed the presence of small aggregates, suggesting that microgel structure can be disrupted at low interfacial loadings. The ability of β-lactoglobulin microgels to form highly elastic interfacial layers may enable improvements in the colloidal stability of food, pharmaceutical and cosmetic products in addition to applications in controlled release and flavor delivery systems.

Electrostatic Stabilization of β-lactoglobulin Fibrils at Increased pH with Cationic Polymers

In order to improve the stability of β-lactoglobulin fibrils formed in acidic conditions to increased pH values (pH 3-7), formation of electrostatic complexes between fibrils and cationic polymers chitosan (CH), amine-terminated poly(ethylene glycol) (APEG), low molecular weight poly(ethylenimine) (LPEI), and high molecular weight poly(ethylenimine) (HPEI) was investigated by electrophoretic mobility, turbidimetry, and atomic force microscopy. Except for suspensions with APEG, addition of polycations increased ζ-potential values of the fibrils at pH 5, 6, and 7, verifying their interactions with fibrils. Maximal increase in ζ-potential at pH 7, indicating optimal electrostatic interactivity, occurred at concentrations (w/w) of 0.05, 0.01, and 0.01% (corresponding to 6.9, 50, and 4 μmol·kg(-1)) for CH, LPEI, and HPEI, respectively. Turbidity of fibril solutions at pH 5, indicating isoelectric instability, was decreased significantly with increasing concentration of CH, LPEI, and BPEI, but not with added APEG. Turbidity was increased at pH 7 with added polycation, except for suspensions containing ≥0.02% HPEI. Fibril length and resistance to aggregation, as observed by atomic force microscopy, were increased at pH 5 with increasing concentration of CH and LPEI, yet only HPEI was capable of maintaining the morphology of fibrils at pH 7. Calculated persistence lengths of the fibrils, as compared to pure fibrils at pH 3 (∼4 μm), were only slightly reduced at pH 5 with CH and at pH 7 with HPEI, but increased at pH 5 with LPEI and HPEI. Improvement in the stability of β-lactoglobulin fibrils at higher pH conditions with the addition of polycations will contribute to their potential utilization in packaging, food, and pharmaceutical applications.

Control of thermal fabrication and size of β-lactoglobulin-based microgels and their potential applications.

HYPOTHESIS Factors influencing fabrication and size of microgels formed from β-lactoglobulin with or without pectin can tune selected attributes for material applications. Protein aggregation was expected to be influenced by pH, added anions, and reducing agents, while ionic strength was expected to be more influenced by electrostatically interacting pectin. EXPERIMENTS Turbidity measurements during thermal aggregation to form microgels were determined for pure β-lactoglobulin as a function of pH, added ionic strength, anion type (chloride, sulfate, and thiocyanate), and reducing agent concentration. β-lactoglobulin and pectin complexation pH values and thermal aggregation were determined by turbidity measurements with added potassium chloride, sulfate, and thiocyanate. Microgel size and morphology were determined by light scattering and atomic force microscopy, respectively. FINDINGS Thermal aggregation of pure β-lactoglobulin increased with decreased pH, reducing conditions, and increased ionic strength with no observed anion effect. β-lactoglobulin microgel radii increased from 86 to 115nm with decreasing pH and increased to 124nm in reducing conditions, while salts promoted agglomeration. Increased ionic strength (0-100mmol/kg) decreased β-lactoglobulin-pectin complexation pH from 5.40 to 5.00, while first increasing and then decreasing thermal aggregation. Thermal aggregation and microgel size were greatest with potassium thiocyanate, followed by potassium chloride and potassium sulfate.

Chitosan-coated amyloid fibrils increase adipogenesis of mesenchymal stem cells

Mesenchymal stem cells (MSCs) have the potential to revolutionize medicine due to their ability to differentiate into specific lineages for targeted tissue repair. Development of materials and cell culture platforms that improve differentiation of either autologous or allogenic stem cell sources into specific lineages would enhance clinical utilization of MCSs. In this study, nanoscale amyloid fibrils were evaluated as substrate materials to encourage viability, proliferation, multipotency, and differentiation of MSCs. Fibrils assembled from the proteins lysozyme or β-lactoglobulin, with and without chitosan coatings, were deposited on planar mica surfaces. MSCs were cultured and differentiated on fibril-covered surfaces, as well as on unstructured controls and tissue culture plastic. Expression of CD44 and CD90 proteins indicated that multipotency was maintained for all fibrils, and osteogenic differentiation was similarly comparable among all tested materials. MSCs grown for 7days on fibril-covered surfaces favored multicellular spheroid formation and demonstrated a >75% increase in adipogenesis compared to tissue culture plastic controls, although this benefit could only be achieved if MSCs were transferred to TCP for the final differentiation step. The largest spheroids and greatest tendency to undergo adipogenesis was evidenced among MSCs grown on fibrils coated with the positively-charged polysaccharide chitosan, suggesting that spheroid formation is prompted by both topography and cell-surface interactivity and that there is a connection between multicellular spheroid formation and adipogenesis.

Influence of PEGylation on the ability of carboxymethyl-dextran to form complexes with α-lactalbumin.

Electrostatic interactions between α-lactalbumin (α-lac) and carboxymethyldextran (CMD) in acidic solutions lead to phase-separated complexes. By adding a non-ionic poly(ethylene glycol) (PEG) chain onto the reducing end of CMD, forming carboxymethyl-dextran-block-poly(ethylene glycol) (CMD-b-PEG), the PEG block was hypothesized to reduce interactions with α-lac and promote formation of a micelle-like complex structure. Formation of complexes between α-lac and CMD-b-PEG or α-lac and CMD was determined following acidification by light scattering and electrophoretic mobility. Phase separation, size, and structure of α-lac/CMD-b-PEG complexes were characterized by turbidimetry, dynamic light scattering, and electron microscopy, respectively. Complexes of α-lac/CMD-b-PEG formed at pH values near pH 6, while α-lac/CMD complexes formed at pH 5.5. Both CMD and CMD-b-PEG decreased the charge of α-lac below pH 5.5 and led to phase separation below pH 5. Shift in charge and the critical pH of phase separation were both sensitive to the α-lac to CMD ratio, while the relative amount of CMD-b-PEG did not significantly influence either. Hydrodynamic radii of α-lac/CMD-b-PEG complexes was between 11 and 20 nm, which increased with increasing α-lac to CMD-b-PEG ratio and with decreasing pH. Spheroidal structures of ∼10 nm were also observed in micrographs that were attributed to α-lac/CMD-b-PEG complexes.

Influence of sulphate, chloride, and thiocyanate salts on formation of β-lactoglobulin-pectin microgels.

Effects of sulphate, chloride, and thiocyanate salts on the heat-induced formation of protein-based microgels from β-lactoglobulin-pectin complexes were determined as a function of pH and protein-to-polysaccharide ratio. Aggregation temperatures were initially decreased at low ionic strength due to shielding of electrostatic interactions between β-lactoglobulin and pectin but increased with further increases in ionic strength. Turbidity of heated mixtures and associated sizes of formed microgels were increased with up to 75 mmol kg(-1) ionic strength. Aggregation and microgel formation were relatively increased in the presence of thiocyanate salts compared to chloride salts and relatively decreased in the presence of sulphate salts, indicating that the inverse Hofmeister series was relevant in this system. Topographical analysis of dried microgels by atomic force microscopy verified that microgels were smallest in the presence of sulphate salts and showed that added ions, particularly thiocyanate, increased the deformability of microgels during drying.

Effect of crosslinking on the physical and chemical properties of β-lactoglobulin (Blg) microgels

HYPOTHESIS Microgels assembled from the protein β-lactoglobulin are colloidal structures with potential applications in food materials. Modifying the internal crosslinking within these microgels using enzymatic or chemical treatments should affect dissolution, swelling, and viscous attributes under strongly solvating conditions. EXPERIMENTS Microgels were treated with citric acid, glutaraldehyde and transglutaminase to induce cross-linking or with tris(2-carboxyethyl)phosphine to reduce disulfide linkages. Change in hydrodynamic particle size due to acidic pH, alkaline pH, ionic strength, osmolyte concentration, ethanol, urea, sodium dodecyl sulfate, and reducing agents was evaluated by light scattering measurements. Changes in microgel nanomechanical properties were evaluated via force spectroscopic measurements in water. FINDINGS Average microgel size increased ∼20% in alkaline pH and with ethanol contents above 10%, and decreased ∼20% with sucrose contents above 10%. Cross-linking by glutaraldehyde and transglutaminase prevented size increases in alkaline pH. Microgel plasticity and elastic modulus were unaffected by treatments. Microgels treated with glutaraldehyde were found to have much greater stability to urea, sodium dodecyl sulfate, and reducing agents when compared to other samples. Even without cross-linking, microgels remained stable against precipitation and dissolution over a wide range conditions, indicating their broad utility as colloidal stabilizers, texture modifiers or controlled release agents in food or other applications.