Innocent B. Bekard

The effects of shear flow on protein structure and function.

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.

Tyrosine Autofluorescence as a Measure of Bovine Insulin Fibrillation

The traditional approach to investigating the partial unfolding and fibrillation of insulin, and proteins at large, has involved use of the dyes 1-anilinonaphthalene-8-sulphonic acid (ANS) and Thioflavin T (ThT), respectively. We compare the kinetic profiles of ThT, ANS, light scattering, and intrinsic Tyr fluorescence during insulin fibrillation. The data reveal that the sequence of structural changes (dimers --> monomers --> partially unfolded monomers --> oligomeric aggregates --> fibrils) accompanying insulin fibrillation can be detected directly using intrinsic Tyr fluorescence. The results indicate that at least two distinguishable structural intermediates precede fibril development. There is no evidence of tyrosinate or dityrosine during insulin aggregation. Obtaining such critical information from the protein itself is complementary to existing aggregation probes and affords the advantage of directly examining structural changes that occur at the molecular level, providing concrete details of the early events preceding fibrillation.

Shear-induced deformation of bovine insulin in Couette flow.

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.

Shear Flow Induced Changes in Apolipoprotein C-II Conformation and Amyloid Fibril Formation

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.

Bovine serum albumin unfolds in Couette flow

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.

α-Helix unfolding in simple shear flow

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 (tc) 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.

A generic class of amyloid fibril inhibitors

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 Alzheimers disease, Parkinsons 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.

Effect of natural biopolymers on amyloid fibril formation and morphology

Amyloid fibrils are associated with the pathogenesis of protein misfolding diseases such as Alzheimers disease. These fibrils typically exhibit different morphologies when grown in vitro, and this has been known to affect their biological properties and cytotoxicity. The formation kinetics and resultant morphology of fibrils formed from the model proteins Bovine Insulin and Hen Egg White Lysozyme have been measured. We show that the presence of gum arabic and pectin during fibril formation cause the amyloid fibrils formed to associate into higher order fibrillar aggregates. It is postulated that the carbohydrates act as a template to promote inter-fibril association, resulting in larger, thicker fibrils. This observation provides some insight into the differences in growing amyloid fibrils in vitro in the absence or presence of other high molecular weight compounds. Furthermore, these findings suggest a method of tailoring fibril structure for applications in nanotechnology and bio-template applications.

The Effect of Shear Flow on Amyloid Fibril Formation and Morphology

Abstract Here we review the effects of shear flow exposure on protein misfolding and fibril formation. The structure and morphology of fibrils formed for a number of model proteins exposed to shear are presented. The effect of shear flow has been shown to be two-fold. Firstly, the hydrodynamic forces cause the proteins to unfold. This is an area of debate in the literature as theoretically the observed effects occur at Peclet numbers which are typically too low to expect any effect. Real-time CD measurements of protein solutions in flow show that the misfolding event involves tertiary structural change followed by changes in the secondary structure. The α-helical content is observed to reduce with a commensurate increase in random chain and β-sheet. The β-sheets then aggregate to form fibrils. The second effect is to cause morphologic changes in the fibrils formed. Fibrils formed in simple Couette flow differ in their structure from those formed in the varying shear fields that occur in typical agitation processes such as shaking and stirring. We conclude that the effect of shear on the fibril formation rate and morphology is of significance to physiology and has potential implications for the cytotoxicity of the fibrils formed. While shear is seen to be important, the true causality for fibril formation in the associated diseases is not fully understood.