Innocent B. Bekard

Amyloid fibrils are large ordered fibrillar aggregates formed from mis-folded proteins. A number of human diseases are linked to the presence of amyloid deposits, including Alzheimer's disease, Parkinson's disease and type II diabetes. One therapeutic strategy for treating amyloid related diseases involves inhibiting fibril formation. Amyloid fibrils are β-sheet rich fibrillar aggregates that associate through hydrophobic interactions between precursor units. In this study, these generic physical properties of amyloid fibrils have been exploited to design a universal class of amphiphilic macromolecular inhibitors. A naturally occurring macromolecule of this structure is arabinogalactan protein (AGP), a component of gum arabic (GA). In addition, two synthetic polymers based on the proposed amphiphilic structure were synthesized and tested. These synthetic mimics, referred to as poly(norbornene glucose ester) (PNGE) and poly(norbornene gluconamide) (PNGA), possess hydrophobic polynorbornene backbones and pendent hydrophilic cyclic and open-chain glucose units, respectively. AGP, PNGE and PNGA all show inhibitory effects on in vitro amyloid fibril formation in bovine insulin (BI), hen egg white lysozyme (HEWL) and amyloid beta 1–40 (Aβ) proteins. Circular dichroism (CD) spectra of the proteins in the presence of the inhibitors suggests that amyloid fibril formation is inhibited by stabilization of the native α-helices of the proteins, as well as binding of the inhibitors to the β-sheet precursors. Based upon these results, glycosylated hydrophobic macromolecules are identified as a promising class of therapeutic agents for amyloid related diseases. Furthermore, we have determined that the intensity of the fluorescent probe thioflavin T (ThT) is dependent on both fibril morphology and the presence of the inhibitors, and is therefore not a quantitative measure of protein conversion to fibrils.

The effect of a low strength oscillating electric field on the conformation of Bovine Serum Albumin (BSA) and Lysozyme in solution has been measured. A purpose built cell has been used to measure the real time autofluorescence and Circular Dichroism of the protein solutions exposed to electric fields of differing strength and frequency. Exposure to the electric fields results in protein unfolding for both Lysozyme and BSA. The applied field strengths are extremely small compared to the protein inter-chain intra-molecular forces. We propose a model whereby the electrophoretic motion of the proteins leads to a frictional force that results in protein unfolding. For BSA and Lysozyme in the electric fields used in this study, the shear rates at the protein surface under electrophoretic motion are of order 10³ and 10⁴ s⁻¹ respectively. Prolonged electric field exposure results in significant frictional energy dissipation in the proteins. The energy dissipated in the proteins results in protein unfolding, which is a critical initial step for protein aggregation and potentially amyloid fibril formation.

Direct measurements of the effect of hydrodynamic forces generated in Couette flow on the secondary and tertiary structure of bovine serum albumin (BSA) are reported. Real time measurements were made using both fluorescence and circular dichroism spectroscopy as complementary measures of protein structure. Our results demonstrate that BSA, a medium sized, predominantly α-helical protein, unfolds irreversibly in simple shear flow. The shear rates used in this study are comparable to those encountered in bioprocessing and in physiology. Flow-induced unfolding of BSA occurs in simple Couette flow where both exposure time and shear rate increase the degree of irreversible unfolding.

Protein molecules are subjected to potentially denaturing fluid shear forces during processing and in circulation in the body. These complex molecules, involved in numerous biological functions and reactions, can be significantly impaired by molecular damage. There have been many studies on the effects of hydrodynamic shear forces on protein structure and function. These studies are reviewed and the implications to bioprocessing and pathophysiology of certain diseases are discussed. © 2011 Wiley Periodicals, Inc. Biopolymers 95: 733‐745, 2011.

The misfolding and self-assembly of proteins into amyloid fibrils that occur in several debilitating diseases are affected by a variety of environmental factors, including mechanical factors associated with shear flow. We examined the effects of shear flow on amyloid fibril formation by human apolipoprotein C-II (apoC-II). Shear fields (150, 300, and 500 s^–1) accelerated the rate of apoC-II fibril formation (1 mg/mL) approximately 5–10-fold. Fibrils produced at shear rates of 150 and 300 s^–1 were similar to the twisted ribbon fibrils formed in the absence of shear, while at 500 s^–1, tangled ropelike structures were observed. The mechanism of the shear-induced acceleration of amyloid fibril formation was investigated at low apoC-II concentrations (50 μg/mL) where fibril formation does not occur. Circular dichroism and tryptophan fluorescence indicated that shear induced an irreversible change in apoC-II secondary structure. Fluorescence resonance energy transfer experiments using the single tryptophan residue in apoC-II as the donor and covalently attached acceptors showed that shear flow increased the distance between the donor and acceptor molecules. Shear-induced higher-order oligomeric species were identified by sedimentation velocity experiments using fluorescence detection, while fibril seeding experiments showed that species formed during shear flow are on the fibril formation pathway. These studies suggest that physiological shear flow conditions and conditions experienced during protein manufacturing can exert significant effects on protein conformation, leading to protein misfolding, aggregation, and amyloid fibril formation.

The unfolding dynamics of the α-helical poly-L-lysine (α-PLL) in Couette flow is reported. Real-time circular dichroism measurements for a range of molecular weights and shear rates have been made. The PLL molecules show a time- and shear rate-dependent unfolding in simple shear flow with a critical strain (t_c) value of ∼10⁵. This strain value is found to be independent of the chain-length of the α-helices. The extent of unfolding is less pronounced with increasing molecular weight (M) for a given strain, showing a linear dependence of the remaining helix, α, on M: α ≈ M. Furthermore, the helix content, α, is found to show a power law dependence with strain: α ≈ (t)^{−1 /2}. A shear-induced rapid unfolding of short chain α-PLL molecules in the flow field occurs. The shear-stability of the larger molecular weights is due to the cohesive forces stabilizing the helix, combined with the associated hydrodynamic screening of helical segments from the full effect of the drag in the flow field. The data are compared with recent molecular dynamics simulations of the dynamics of dilute polymer solutions in shear flow and scaling arguments are used to interpret the trends in the data.

We have applied a uniform, shear-driven flow field (Couette flow) to study the effect of shear on the structure and conformation of aqueous bovine insulin, in situ and in real time, using intrinsic Tyr fluorescence and circular dichroism (CD) spectroscopy. The morphology of post-shear insulin samples was analyzed using atomic force microscopy (AFM). Both fluorescence and CD data show a shear-dependent deformation of bovine insulin in Couette flow. The shear effect is more pronounced with increasing shear rate. AFM images show large aggregates for insulin samples sheared at 200 and 400 s⁻¹, whereas samples sheared at 600 s⁻¹ contained fibrillar forms. We hypothesize that helical segments unfold upon extensional strain in the deformation flow field, resulting in unstructured, aggregation-prone insulin molecules. The occurrence of rotational diffusion in the direction of flow facilitates the coalescence of deformed insulin molecules into oligomeric aggregates. The size of the insulin aggregates diminished with increasing shear rate. This shows that the deformation cycle in fast flow fields retards the formation of large aggregates and promotes the ordering of deformed insulin molecules into the more stable fibrillar forms.