ReviewThe Effects of Shear Flow on Protein Structure and Function

Innocent B. Bekard,1 Peter Asimakis,1 Joseph Bertolini,2 Dave E. Dunstan11 Department of Chemical and Biomolecular Engineering, University of Melbourne, Parkville, VIC 3010, Australia

2 Research and Development Department, CSL Biotherapies, 189-209 Camp Rd, Broadmeadows, VIC 3047, Australia

Received 14 February 2011; revised 20 April 2011; accepted 26 April 2011

Published online 4 May 2011 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/bip.21646

This article was originally published online as an accepted

preprint. The Published Online date corresponds to the

preprint version. You can request a copy of the preprint by

emailing the Biopolymers editorial office at biopolymers@wiley.

com

INTRODUCTION

Proteins are linear polymer chains of combinations of

the 20 amino acid residues. These complex macro-

molecules form an essential part of an array of bio-

logical processes in living systems, such as structural

components of cells, biological catalysts (enzymes),

and chemical messengers (hormones). The functional prop-

erties of proteins depend on their unique three-dimensional

structure which is determined primarily by the amino acid

sequence of individual chains. The energy threshold between

the native and denatured state of a protein is low, thus the

physicochemical environment is a critical determinant of

protein structure and function.1 Perturbation of the native

protein conformation results in loss of catalytic activity or

biological function,2,3 and in many cases aggregation and

amyloid bril formation.46

Structural perturbation of protein molecules occurs as a

result of conditions of temperature, pH, and ionic strength

as well as through the presence of denaturants (e.g., urea,

guanidine), organic solvents, and surfactants.7 In addition,

structural destabilization can result from exposure to hydro-

dynamic shear forces810 originating from shaking, sonica-

tion, mixing, vortexing, and ow through conduits.7,11,12

The effect of uid shear stress on protein structure is a

subject of practical interest because it is a common pheno-

menon in bioprocessing.13 Proteins have been shown to

exhibit conformational dynamics in shear ow.1417 Protein

solutions are exposed to shear stresses during bioprocessing

steps as a result of centrifugation, fractionation, pumping,

and ultraltration.13,18,19 Similarly, the shipping and

handling of biotherapeutics and protein based bioprocessing

reagents such as monoclonal antibodies, hormones, cyto-

kines, and enzymes can result in signicant agitation. For

these reasons, an understanding of the effects of shear ow

on protein stability, and a means of testing and measuring

this effect, would allow the appropriate design and selection

of manufacturing processes, conditions, and formulations

which would ensure maximum yield and stability.

It has been known for some time that considerable shear

stress is generated by blood ow in the circulatory system.20

However the pathophysiological implications of this have not

been explored. Thus, this review will also examine relevant

papers in this area and highlight implications to the patho-

genesis of some diseases.

ReviewThe Effects of Shear Flow on Protein Structure and Function

Correspondence to: Dave E. Dunstan; e-mail: davided@unimelb.edu.au

Innocent B. Bekard and Peter Asimakis contributed equally to this work.

ABSTRACT:

Protein molecules are subjected to potentially denaturing

uid shear forces during processing and in circulation in

the body. These complex molecules, involved in numerous

biological functions and reactions, can be signicantly

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: 733745, 2011.

Keywords: shear ow; protein; unfolding; aggregation;

amyloid bril; physiology; bioprocessing

VVC 2011 Wiley Periodicals, Inc.

Biopolymers Volume 95 / Number 11 733

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Studying the Effect of Shear Flow on ProteinsExtensional Flow vs. Simple Shear Flow. The common ow

elds applied in shear studies are extensional ow and simple

shear ow. A homogeneous extensional ow, also called

elongational ow or stretching ow, is characterized by a lin-

ear velocity gradient of the form vy _cy along the directionof ow (Figure 1).21 The strain rate, _c @xy@y , is constant. Anexample is the ow of a polymer solution through a con-

verging channel with hyperbolically-shaped walls. This

should not be confused with well developed ow of an

incompressible uid through a circular pipe of innite

length, which is characterized by inhomogeneous shear.

Here, the ow pattern is of the Poiseulle type, showing a

parabolic velocity prole where both the shear rate and

uid velocity varies as a function of the distance from the

vessel boundary (Figure 1). The strain rate is given as

_c 2y0a where y0 is the distance between the center of thepolymer and the ow axis and a is a measure of the curvatureof the ow prole.22

Simple shear ow on the other hand describes uid ow

characterized by a velocity gradient perpendicular to the ow

eld (Figure 1). It is modeled as a linear superposition of

rotational ow with vorticity x, and elongational ow with

strain rate _c @vx@y .16 In the rotational component of the oweld, protein molecules experience whole body rotation

with no hydrodynamic shear strain, hence maintain their

structural integrity. However, protein molecules are sub-

jected to stretching events when oriented in the extensional

ow eld. The hydrodynamic shear stress resulting from

these events is thought to destabilize the native protein

structure, leading to aggregate formation.17 For Newtonian

uids, the mathematical relationship between shear stress

(s) and velocity gradient ( _c) in a given ow eld is expressedas s g _c.23 A tangential stress of this type is called shearing,where innitesimally thin layers of uid slide over each other

in laminar ow. Shear stress has units of force per unit area

(N/m2), and is a measure of the actual hydrodynamic drag

forces acting on the protein solution under ow. The velocity

gradient, also referred to as the shear rate, has units of inverse

time (s21).

It is noteworthy that the magnitudes of rotational and

elongational components of simple shear ow are equal (|x|5 | _c|). This implies that the conformational dynamics ofprotein molecules in the ow eld are dictated by the ran-

dom occurrence of both events. In practice, the rotational

and elongational components of ow differ in magnitude.

FIGURE 1 Schematics of the common velocity proles in ow. (A) Homogeneous extensional

ow typically associated with cross-slot geometries. The shear rate is constant. (B) Well developed

ow in a conduit. The velocity prole is parabolic, typical of Poiseuille ow. In addition, the uid

velocity is maximal at the center of the tube, approaching zero at the boundaries. The converse is

true for the uid shear rate. (C) Simple shear ow, showing a velocity gradient perpendicular to

the direction of ow. In all cases (AC), the arrows show both the direction of ow as well as the

velocity prole.

734 Bekard et al.

Biopolymers

Devices for Generating Shear Flow

In many protein studies, shear ow is generated by subjecting

protein solutions to ow patterns characterized by uniform or

heterogeneous velocity gradients.18 Shear devices that create a

near uniform velocity gradient through protein solutions pro-

vide a well controlled and quantiable shear stress. Hence,

such devices are ideal for studying the effects of shear forces on

protein molecules. Conversely, heterogeneous velocity gra-

dients, for example, resulting from stirring or shaking, provide

poorly controlled shear conditions that are difcult to quantify.

It is worth noting that the differences in experimental devices

applied in shear studies, and the associated data interpretation,

could be a major contributory factor to the inconsistent exper-

imental results reported throughout the literature.

Several studies that show the effect of shear ow on a number

of proteins, along with the shear devices employed, are summar-

ized in Table I. By virtue of the differences in experimental design,

the shear (strain history) as well as the shear stress values, which

are not given here, varies signicantly among the different experi-

ments. The experimental ow devices used for protein studies

fall into two main categories: capillary and rotational devices.

Capillary/Microuidic Flow Devices

Capillary ow is where a uid is forced through a conduit of

known dimensions, by applying a given pressure difference

between the inlet and outlet of the conduit.43 Here, the uid

velocity prole is of the Poiseuille type (non-homogeneous

shear ow) with the maximum shear rate at the uid-vessel

boundary, reducing gradually towards the vessel center

(Figure 1B). Very high shear rates, up to 105 s21, can be

achieved but over short residence times on the order of

milliseconds.41,44 A schematic of a typical capillary ow tube

is shown in Figure 2. It has been reported that the short resi-

dence time spent in the ow eld could contribute to

incomplete protein stretching (unfolding).45 However, recir-

culation of protein solutions through conduits have been

successfully applied to increase residence times,32 offering a

good model system for uid shear stress in ultraltration

units and membranes.

Rotational Flow Devices

Cone and plate, parallel plate and concentric cylinder visc-

ometers are three types of rotational ow devices commonly

Table I Studies of the Shear-Stability of Protein Systems Using a Variety of Shear Devices

Device Geometry Protein Shear Rate/Rotational Speed Result References

Couette Insulin 200 s21 Unfolding and aggregation 14

Poly-L-lysine [333 s21 Unfolding 24b-lactoglobulin 25 s21, 150 s21 Fibril formation 25, 26von Willebrand factor \3.0 3 103 s21 Unfolding 27Amyloid-b 50 s21 Fibril formation 28

Taylor-Couette Lysozyme 750 rpm,[14 s21 Unfolding 29, 30Cytochrome-c 750 rpm No change 29

Cone and plate a-amylase 120 s21 Loss of activity 31Fibrinogen 290 s21 Degradation 32

Catalase 91.5 s21 Loss of activity 33

Rennet 9.15 s21 Loss of activity 33

Glycoprotein Ib and IIb-IIIa 8.23 103 s21 Unfolding 34von Willebrand factor 6.7 3 103 s21 Aggregation 35

Concentric cylinder Urease 683 s21 Particle formation 36

Catalase 683 s21 Particle formation 36

Alcohol dehydrogenase 683 s21 Particle formation 37

Deoxyribonuclease 1.5 3 103 rpm No change 38Growth hormone 1.5 3 103 rpm Unfolding and fragmentation 38Urease 48 s21 Loss of activity 39

Capillary Catalase 67 s21 Loss of activity 40

Catalase 4.6 3 104 s21 Minor loss of activity 36Cytochrome-c 2.0 3 105 s21 No change 41

Microuidic cell von Willebrand factor [13 103 s21 Unfolding 42

FIGURE 2 Schematic of a simple extrusion glass capillary vis-

cometer. The arrows show the direction of ow.

Effects of Shear Flow on Protein Structure and Function 735

Biopolymers

used to impart shear stress on protein systems across a nar-

row gap (Figure 3). All models consist of two parts where

one rotates with the other being stationary. In the case of the

concentric cylinder, also known as Couette or coaxial cylin-

der ow-cell, there are several sorts that differ in basic design

but operate on the same principles.46 However, a through-

cell Couette arrangement is ideal for generating a simple

shear ow, as the design minimizes any end effects.47 This

type of device also curtails particle settling or migration away

from the solid boundaries, thereby limiting variations of

uid properties across the gap.48 In addition, a nominal

shear rate can be calculated for each rotational speed. At high

rotational speeds, large deviations from the ideal ow may

occur due to the formation of vortices. It is recognized that

vortices occur in extensional ow regions in uids, and

should therefore be avoided if a simple shear ow is

desired.49 The main advantage of rotational shear devices

over that of the capillary design is that the entire test solution

is sheared at a near constant rate. This permits the study of

any time-dependent behavior of protein samples over an

extended period of time. In the through-cell Couette

arrangement, the shear rate can be regarded as constant only

if the gap width is much less than the radius of the inner cyl-

inder.50 In the cone-and-plate design, the solution is con-

tained in the space between a cone of large apex angle and a

plate with a at surface normal to the axis. For small angles

between the cone and plate, the shear rate is considered con-

stant for all radial positions.

The Effects of Shear Flow on Protein SolutionsProtein Function. Preliminary studies on the relationship

between shear ow and protein structure exploited the cata-

lytic activity of enzyme solutions as a means of measuring

protein integrity. Thus, a decrease in enzyme activity implied

a structural deformation of the native enzyme molecules.

These early studies were critical in dening the appropriate,

and operation of, devices to allow the study of the behavior

of proteins in shear ow, allowing the estimation of shear

forces required to perturb protein function with insight into

possible resulting structural changes.

Studies on catalase, rennet, and carboxypeptidase solu-

tions sheared in a narrow gap coaxial viscometer40 at shear

rates between 9.15 and 1155 s21 for 90 min, showed a strong

correlation between decrease in enzyme activity and shear

(shear rate 3 time). A loss of enzyme activity occurred for

shear greater than 104 although the rennet solutions partially

regained activity upon the cessation of shearing. The

observed enzyme inactivation was attributed to the breaking

of the molecules tertiary structure when oriented appropri-

ately in the shear eld. The authors discounted any signi-

cant contribution from the airliquid interface in the

viscometer, where protein molecules, by virtue of their

amphipathic nature, may aggregate (causing inactivity). It is

also possible that the loss in enzyme activity was due to a

ow-induced limited residence time for enzyme-substrate

interaction. A similar shear-effect was observed upon expo-

sure of catalase and rennet solutions to shear rates in the

region of 104 s21 in model ultraltration systems.33 Whereas

the catalase solutions lost some activity during ultraltration,

the rennet solutions did not suffer inactivation in the circula-

tion lter systems. Further studies on plasma brinogen,

sheared in a Weissenberg rheogoniometer (shear rates 290

1155 s21) with a Couette attachment, revealed a tempera-

ture-independent loss in its clottability.32

Studies on the shear-stability of urease, using a coaxial cyl-

inder viscometer, which allowed direct monitoring of urease

catalyzed hydrolysis of urea during shear exposure, showed

continuous decrease in the rate of urease activity with

increasing shear rate (48, 288, 741, and 1717 s21).39 The par-

tial deactivation of urease revealed both reversible and irre-

versible elements. The reversible inactivation was ascribed to

hydrodynamic distortion of the enzymes structure in ow,

whereas permanent inactivation was attributed to the break-

ing of the enzymes tertiary structure. The permanent loss in

urease activity correlated with shear strain, with a critical

shear value of 105, similar to that previously reported.40 A

comparison of catalase degradation in streamline and turbu-

lent ow regimes, upon mixing in a cylindrical container,

showed a signicant inactivation in the latter where a vortex

and air bubbles had formed in the solution.51 This suggested

that the airliquid interface may have contributed signi-

cantly to catalase degradation. It was also shown that shear

stress in selected microltration membranes caused a signi-

cant decrease in the catalytic activity of yeast alcohol dehy-

drogenase (ADH), under solution conditions where the

FIGURE 3 Schematics of the common rotational ow devices:

(A) concentric cylinder, (B) cone and plate, and (C) parallel plate

rheometers.

736 Bekard et al.

Biopolymers

enzyme was slightly unstable.52 This observation was attrib-

uted to membrane-enzyme interactions resulting from a

shear-induced deformation of the enzyme structure.

In studies of the shear-stability of ADH solutions using a

high shear concentric viscometer, a rotating disk reactor and

several pumps, it was found that fully active tetrameric mole-

cules of ADH showed negligible change in activity even for

strain rates as high as 26, 000 s21 over 1 h in the high shear

device.53 Turbulent regimes in the rotating-disk reactor

(3600 rpm for 5 h) and up to about 1500 passes in the

various pumps likewise had little effect on ADH activity.

However, the introduction of a gas-liquid interface in the

rotating-disk reactor resulted in a 40% decrease in the initial

ADH activity after 5 h. Similarly, a signicant decrease in

ADH activity was observed in both a gear and a Jabsco pump

after 2000 passes.53 This report suggested that interfacial

denaturation, and not shear per se, accounts for the defor-

mation of proteins in solutions exposed to shear ow, and

that the native conformation of globular proteins, when in a

chemically stable environment, are stable in shear ows usu-

ally encountered in practice.

The importance of an airliquid interface in promoting

shear-induced protein denaturation was shown in a study

with progesterone 11a-hydroxylase complex, using anopen concentric cylinder viscometer and a horizontal vis-

cometer closed at both ends.54 It was observed that the

gas-liquid interface, in concert with high shear regimes,

which replenished the protein at the interface, promoted

rapid protein denaturation as previously reported.53 In

addition, a wall surface effect on the enzyme complex was

observed as the gap width of the viscometer was reduced

from 0.475 to 0.15 mm. Furthermore, it was observed

that viscous enzyme solutions showed a marked decrease

in activity as a function of shear stress.54 These observa-

tions lead the authors to propose that the applied shear

stress resulted in enzyme denaturation via either the rup-

ture of the enzyme membrane or the enhanced solubiliza-

tion of component(s) of the enzyme complex by the ve-

locity gradient in the agitated solution.

The above studies clearly showed that shear could have a

profound effect on the activity of enzymes, and by inference

on protein structure. The shear-effect was more pronounced

in the presence of an airliquid interface. A key limitation of

the above studies was that any deduction of structural change

was based on indirect evidence derived from enzyme activity.

Indeed, whereas shear may not have caused signicant struc-

tural damage, a decrease in enzyme activity could result from

minor modications to an enzymes active site. In many of

the experiments, the enzymes were subjected to intermittent

shearing force to allow sampling. Therefore, signicant time

would have elapsed between sampling of solutions and test-

ing for enzyme activity. This would have allowed reversible

structural changes to revert and thus go undetected. The

advent of new experimental techniques, allowing real-time

measurements of the solution conformation of proteins, is

providing new insight into the shear-stability of proteins

with respect to variations in the sensitivity of different pro-

teins to shear, and the specic molecular changes occurring

in response to shear exposure.14,55

Protein Structure

Human von Willebrand Factor (vWF), a blood plasma pro-

tein with important functions in coagulation, was amongst

the rst of protein systems, besides enzymes, to be studied

for shear induced structural changes. vWF molecules, which

are initially secreted by the endothelial cells as large, hyper-

active multimers, with relative molecular mass up to 50 3106, circulate as a series of smaller, less active proteolytic frag-

ments in the plasma as a result of the action of the metallo-

protease ADAMTS-13.56,57 Interestingly, the proteolysis of

large vWF multimers is not observed when normal plasma is

incubated in vitro.58 This lead to the conclusion that the gen-

eration of proteolytic fragments of vWF in circulation

depends on haemodynamic shear stress.57 The size distribu-

tion of vWF multimers in microcirculation is critical to its

haemostatic potential.

Tsai et al. investigated this theory by perfusing normal

plasma through long capillary tubes (190 or 354 cm in

length), at shear rates between 1508 and 4761 s21, followed

by an examination of the size distribution of vWF using a

combination of SDS agarose gel electrophoresis and densito-

metric scanning analysis of autoradiographs.59 Relative to

unsheared plasma, an increase in smaller vWF multimers was

observed in the sheared samples at the expense of the larger

multimers, especially in high shear regimes. These observa-

tions lead to the conclusion that shear stress might be

involved in the modulation of the size distribution of vWF in

circulation by enhancing the proteolytic cleavage of large

vWF multimers.59

In a subsequent study by Siedlecki et al., using a combina-

tion of atomic force microscopy and a rotating disk system,

vWF molecules were observed (visually) to undergo a shear-

induced conformational transition from a globular state to

an extended chain conformation at a critical shear stress

value[31.5 dyn/cm2 ( _c 4618.8 s21).60 The result was theexposure of intra-molecular domains of vWF. It was sug-

gested that shear-induced structural changes in vWF may

have an important role in platelet adhesion and thrombus

formation in regions of high shear stress, as would develop at

the site of a bleeding injury.60

Effects of Shear Flow on Protein Structure and Function 737

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To this point, it had been considered that clot formation

was initiated through direct shear activation of platelets.58

This was substantiated in a later study,61 which investigated

the mechanics involved in the formation of reversible tether

bonds between the platelet glycoprotein receptor Iba and theA1 domain of surface-immobilized vWF under hydro-

dynamic shear ow. Puried platelets were perfused over

plasma vWF-A1 coated surfaces in a parallel-plate ow

chamber at shear stresses between 0.2 and 8 dyn/cm2. Platelet

tethering was visualized using a Nikon microscope equipped

with a high speed video camera, and the motion of the teth-

ered platelets calculated using an analytical two-dimensional

model. The results showed that hydrodynamic compressive

forces inuence tether bond formation between platelets and

vWF.61

Recently, uorescence microscopy has been used to

directly visualize individual uorescently labeled vWF multi-

mers under hydrodynamic stress in a microuidic device.42

vWF was uorescently labeled with Alexa uor 488, which

was attached to the primary amines of the glycoprotein using

tetrauorophenyl ester. It was found that shear rates greater

than 1000 s21 triggered a reversible stretching of vWF in so-

lution. Furthermore, real-time measurements of the confor-

mational dynamics of vWF in response to laminar shear ow

in a Couette ow-cell, using a combination of small angle

neutron scattering (SANS) spectroscopy and quantitative

modeling, showed an irreversible conformational change in

vWF, under hydrodynamic shear stress, at shear rates 3000s21.27 The observed conformational change was found to

involve the exposure of hydrophobic domains at shear rates

[2300 s21, as indicated by an increase in the binding afnityof the hydrophobic dye 1,10-bis(anilino)-4-,40-bis(naphta-

lene)-8,80-disulfonate (bis-ANS).27 The shear-effect was

more pronounced with increasing shear rate.

A more recent single-molecule investigation of the shear-

stability of vWF has provided more insight into the relevance

of shear stress in the homeostatic regulation of the size of cir-

culating vWF multimers as well as the potentiation of vWF-

mediated platelet aggregation.62 Specically, the study exam-

ined the folded and unfolded states of a single A2 domain of

vWF, which contains the site of cleavage for ADAMST-13, in

the presence and absence of the metalloprotease. The A2

domain of vWF was unfolded by directly applying force with

laser tweezers. The results showed that only the unfolded A2

domain is cleaved by ADAMST-13.62 These observations cor-

roborated previous studies which found that shear-induced

stretching of large vWF multimers results in the exposure of

the scissile Tyr1605-Met1606 bond within the A2 domain, faci-

litating cleavage by ADAMST-13 in normal plasma.56,57

Smaller vWF multimers in microcirculation equally undergo

stretching in regions of high shear, such as a bleeding arte-

riole, which expose bonding sites in the A1 domain of vWF

for complexation with platelets during haemostasis.57,60,63

While studies of vWF provided clear evidence that shear

ow can alter protein structure, it may be argued that differ-

ent proteins experience different shear since the strain rates

required to trigger structural alterations decreases with

increasing molecular weight and solvent viscosity. For exam-

ple, studies on the effect of high shear rates on ferric equine

cytochrome c, a small globular protein of molecular weight

12,384 Da, showed no signicant structural alterations when

exposed to shear rates as high as 105 s21 in a capillary ow

device.41 By applying a simple model, the authors suggested

that a shear rate of 107 s21 would be required to denaturea small globular protein in water.41 However, the transient

residence time associated with capillary ow devices may

explain the hysteresis observed in such systems.45 More

importantly, the results41 contradict the observed abrupt

stretching of vWF ([20,000 kDa) in a microuidic device ata threshold shear rate of 103 s21.42 Clearly, the larger, more

complex vWF would be expected to be more susceptible to

shear stress relative to cytochrome c. However, it is notew-

orthy that specic conformational characteristics such as

a-helix and b-sheet composition, and intra-molecularinteractions (e.g., hydrophobic, electrostatic) and covalent

bonds (e.g., disulde), would be an important determinant

of protein stability.

Thus, protein systems with comparable molecular weights

may differ in their shear-stability due to differences in their

solution properties. For example, previous studies on

the effects of both high shear ([107) and high shear rate([105 s21), using concentric cylinder-based systems, onrecombinant human growth hormone (rhGH) (22 kDa)and recombinant human deoxyribonuclease (rhDNase)

(31 kDa) showed that rhDNase was more resistant toshear-effects.38 For instance, whereas scanning microcalorim-

etry and SDS-PAGE analysis of extensively sheared solutions

showed changes in the melting temperature of rhGH and the

presence of low molecular weight fragments, respectively, no

such changes were observed for rhDNase. Interestingly, the

observed structural changes in rhGH were not detected in

both the near and the far-UV circular dichroism spectral pro-

le of the sample.38 Further studies on both samples showed

that rhGH denatured upon exposure to high shear in the

presence of an airliquid interface whereas rhDNase

remained relatively stable.64 The stability of rhDNase was

attributed to its comparatively high surface tension and low

foaming tendency in solution.

Intriguingly, it has been shown that human and bovine

albumin samples, which differ in only two amino acids,

738 Bekard et al.

Biopolymers

show different aggregation kinetics upon shaking at high

shear rates ([8000 rpm).65 Multi-angle laser light-scatter-ing measurements showed that in addition to the mono-

mer-dimer transition observed in both albumin samples

upon shearing, human serum albumin also formed trimers

especially at high shear rates. Since the protein concentra-

tion and solventair interfaces were considered as xed

factors, the observed differences in the degree of albumin

aggregation was attributed to the minor variation in their

primary structure.

Protein Aggregation

As has been discussed above, shear can result in protein

denaturation and compromised function. It is also well

established that denaturation of proteins can lead to aggrega-

tion.66 Hence, the use of agitation (uncontrolled shear stress)

to both induce and accelerate protein aggregation and amy-

loid bril formation is common practice throughout the lit-

erature.66 For example, under favorable solution conditions,

various forms of agitation including shaking, sonication and

stirring have been successfully applied to accelerate the aggre-

gation of insulin,6771 amyloid-b,28,72 b2-microglobulin,73,74

b-lactoglobulin,25 albumin,65 and the PDZ domain frommurine Protein Tyrosine Phosphatase Bas-like (PDZ2)75

among others. In one particular study, a range of structurally

diverse protein systems including BSA, myoglobin, lysozyme,

Tm0979, SOD and hisactophin were shown to generate amy-

loid like structures following sonication.76

Shear effects on protein aggregation have also been stud-

ied in uniform ow elds. For example, a shear-induced

nucleation, without ow enhanced aggregation, of preheated

b-lactoglobulin solutions (0.5 wt%) at low pH has beenreported.77 b-lactoglobulin solutions were exposed to Cou-ette ow in a rheometer, at a moderate shear rate of 200 s21

over 5 h, while measuring the ow-induced birefringence.

Thermal treatment of the b-lactoglobulin samples triggeredthe formation of metastable pre-brillar aggregates, without

which bril formation was absent in the sheared samples.

Shearing of b-lactoglobulin samples was done either continu-ously or in short pulses, both producing a similar degree of

bril formation. These observations lead to the conclusion

that brief mechanical perturbation of an aggregation prone

protein solution was enough to enhance bril formation.77

That is, shear ow only triggered the nucleation of bril for-

mation but did not inuence the polymerization of preaggre-

gates into mature brils, discounting orthokinetic coagula-

tion under the experimental conditions. However, the brils

formed from the continuous shear treatment showed a

smaller variance in Gaussian length distribution relative to

those from the pulse shear treatments, indicating a homoge-

nizing effect of the continuous shear treatment. Evidently, a

synergy of heat and agitation triggered rapid bril formation.

Another study showed that the aggregation of preheated

b-lactoglobulin solutions in Couette ow was shear rate de-pendent.25 In addition, if shearing was performed prior to

thermal treatment, a rapid enhancement of brillar species

still occurred, suggesting a shear-induced formation of the

precursor nuclei required for bril development as previously

reported.77 Interestingly, at shear rates[500 s21, bril degra-dation, possibly arising from the persistent stretching (exten-

sional ow) events in the ow eld, was observed. A further

study78 revealed that a sufciently high b-lactoglobulin con-centration ([3 wt%) was required to initiate bril formationwhen samples were heated and sheared simultaneously. In

addition, the amount of brils formed increased as a function

of shear rate up to 337 s21 beyond which an opposing shear

effect, similar to that reported earlier,25 was observed. The

paradoxical shear-effect was explained by a ow enhanced

polymerization of the bril nucleus in low shear regimes,

whereas in high shear regimes, the extensional component of

the ow eld overwhelmed the nascent inter-b-strand hydro-gen bonds stabilizing the aggregating brils. Furthermore, the

authors observed that the shear applied produced shorter

brils and also enhanced, below a critical bril concentration,

the viscosity of the resulting bril solutions.78

More recently, a similar ow enhanced bril formation

has been observed in aqueous solutions of bovine insulin,14

thermally treated amyloid-b28 and b-lactoglobulin.26 Theshear-stability of bovine insulin in Couette ow was probed

directly via novel circular dichroism and tyrosine autouor-

escence measurements, in situ and in real time. For a given

shear rate, helical segments of native insulin were observed to

unfold as a function of time. Atomic force microscopy

images of the sheared samples revealed the presence of bril-

lar forms in relatively strong shear regimes (600 s21).14 The

shear-effect on preheated amyloid-b samples28 exposed toCouette ow showed similar trends to that reported earlier.25

For the b-lactoglobulin studies,26 the authors compared themorphology and mechanical properties of brils formed

under heterogeneous (stirred) and uniform (Couette) ow

conditions using atomic force microscopy. They found that

b-lactoglobulin brils resulting from heterogeneous ow hadtwisted ribbon-like morphologies and higher mechanical

strength relative to the beaded brils formed in Couette ow.

Evidently, the intertwining of brils produced in the variable

shear regime imparted additional mechanical stability to the

overall bril structure. It was hypothesized that polymeriza-

tion of bril seeds occurs in the ow eld, and in the case of

stirred samples, mature brils anneal into twisted ribbons

possibly via orthokinetic coagulation. Taking together, even

Effects of Shear Flow on Protein Structure and Function 739

Biopolymers

if in some cases the initial structural destabilization required

for protein aggregation is not triggered by shear, its ability to

accelerate the polymerization of metastable prebrillar aggre-

gates into mature brils still has grave implications in bio-

processing and physiology.

Regarding the mechanism of shear-induced protein

aggregation, there is a general consensus that mechanical

perturbation of a protein molecule often results in a struc-

tural destabilization of the native conformation, leading to

the exposure of sequestered hydrophobic residues to the

surrounding medium. Solvent-exposed hydrophobic groups

nucleate via hydrophobic interactions and subsequently ag-

gregate. Several studies show that the initial destabilization

of the protein structure occurs at the solventair or sol-

ventsolid interface, where surface tension forces unfold

the native protein.36,37,64,79 The airwater interfacial force

is estimated to be 140 pN,80 taking the airwater interface

to be 2 nm in depth with a 0.07 N/m surface tension.81

This value is comparable to the 150 pN observed to

unfold globular proteins in atomic force microscopy stud-

ies.82 In addition, as agitation increases the turnover of

the airliquid interface, and thus the number of protein

molecules interacting with this interface, the nucleation of

solvent-exposed hydrophobic species via hydrophobic

interactions, and hence the aggregation of the bulk protein

solution, increases dramatically.72 In fact, the inuence of

agitation can be so profound that, in the case of insulin,

it was found to attenuate the effects of other parameters,

such as protein concentration and ionic strength, that

inuence the kinetics of amyloid bril assembly.68

Molecular Models and Theoretical Aspects

The complexity of protein molecules with respect to confor-

mational heterogeneity as well as intra- and intermolecular

interactions between amino acid residues, with the consequent

differences in the exibility of secondary conformations and

tertiary congurations between proteins, results in variations

in their solution properties in shear ow. Indeed, differences in

the levels of association in diverse protein systems dictate the

different degrees of shielding, especially of hydrophobic resi-

dues concealed in the protein matrix, from hydrodynamic drag

during shear ow.83,84 Hence, it is difcult to obtain uniform

data to allow the formulation of a theoretical model relating

protein conformation to shear. For these reasons, dilute solu-

tions of homopolymers, especially unbranched polymer

chains, have been used as model systems for shear studies

because of their inherent structural uniformity.

By virtue of its conformational plasticity in response to

temperature, pH, salt concentration and alcohol content in

solution,24 poly-L-lysine is commonly used as a model system

in protein studies.85 The homopolypeptide exists as a

random coil at neutral pH, a-helix at high alkaline pH andb-sheet at temperatures[308C.86 For these reasons, poly-L-lysine is the ideal choice in probing the initial conformational

transitions, under denaturing conditions, between the three

basic protein secondary conformations namely: random coil,

a-helix, and b-sheet.Experimental studies on the shear-stability of poly-L-ly-

sine solutions (437 kDa), in an initial a-helical conformation,showed an increase in solution viscosity and turbidity with

shearing time.87 A combination of Raman spectroscopy (col-

lected in situ) and circular dichroism were employed for data

acquisition in a Couette ow cell at 150 s21. In addition, a

ow induced-gelation and an increase in the b-sheet contentof the samples was observed in solution concentrations[0.3g/dL. However, the circular dichroism data further revealed a

reversal of the a-helix to b-sheet conformational transitionafter prolonged shearing ([50 min). The observed confor-mational change and subsequent aggregation of the sheared

samples was thought to involve ow-enhanced hydrophobic

interactions between individual poly-L-lysine molecules in

solution.87 Further studies, via real-time circular birefrin-

gence measurements, showed a reversible, shear-induced, he-

lix-to-coil transition of poly-L-lysine in simple shear ow.24

The conformational transition was observed at a critical

strain rate of 300 s21, and the change was attributed to a

shear-induced breakage of intramolecular hydrogen bonds in

a-helical poly-L-lysine.More recently, the molecular-weight-dependence of the

shear-induced unfolding of a-helical poly-L-lysine in Couetteow has been measured in real-time using circular dichroism

spectroscopy.55 The authors observed that the shear-stability of

the helices increased as a function of molecular weight. The hys-

teresis observed in the heavy chains was attributed to the large

network of hydrogen bonds (cohesive force) stabilizing the helix

structure, and the associated hydrodynamic screening of helical

segments from the drag in the ow eld. Interestingly, irrespec-

tive of the molecular weight of poly-L-lysine, unfolding of the

helical segments was found to occur at a critical strain ( _ctc)value of 105 (Figure 4), similar to that observed in globularproteins.39,40 For this reason, the authors proposed that the

shear rate is not as critical as the duration of its application,

which makes the idea of a critical shear rate arbitrary. In

addition, the helix content, a, was found to show a powerlaw dependence with strain: a _ct1=2.

The inherent monodispersity associated with lamda bacte-

riophage DNA made it another ideal model for studies of

polymer dynamics in hydrodynamic ows. For example, a

novel real-time uorescence videomicrocopy of uorescently

labeled single DNA molecules was developed to visualize the

740 Bekard et al.

Biopolymers

conformational dynamics of DNA chains in both elongation21

and shear16 ows. The elongation and shear ow experiments

were performed in a microfabricated ow cell and between

two parallel glass plates (50 lm gap), respectively, at verylow shear rates ranging from 0.05 to 4.0 s21. It was found that,

at relatively high shear rates, the sharp coil-stretch transitions

of DNA chains observed in pure elongation ow was absent in

shear ow.16,21 Individual chains in steady shear ow rather

showed large, aperiodic temporal uctuations in conforma-

tion, consistent with what had been predicted in theory.84 In

addition, stretching of DNA chains in shear ow occurred at a

strain value\50 (101),16 which is several orders of magni-tude lower than the 105 critical strain value observed forpoly-L-lysine55 as well as folded proteins.39,40

Jaspe and Hagen41 suggests that coil-stretch transitions

readily occur in homopolymer systems because the free

energy cost of unfolding is negligible compared to that of a

native protein, and this was purported to explain why an ex-

traordinarily high shear rate (107 s21) is required to destabi-

lize the latter. Another study88 estimated a destabilizing

strain rate in the region of 108 s21 for collapsed polymers in

planar shear ow.

Classical theories on the shear-stability of polymer chains

predict that a polymer chain would remain in a coil state in

shear ow until a critical velocity gradient, which triggers an

abrupt unwinding of the coil, is reached.86 In addition,

abrupt conformational transitions are not expected in simple

shear ow since the occurrence of extensional strain, hence

polymer stretching, is random. It is hypothesized that at very

high strain rates, where polymer chains spend a relative short

time (residence time) in an extensional ow eld, no unfold-

ing or incomplete chain extensions may occur.45 These pre-

dictions were substantiated in later studies that simulated

polymer dynamics in shear ow. For example, in molecular

dynamics (MD) simulations of a grafted collapsed macro-

molecule in strong uniform ow, it was observed that a exi-

ble macromolecule remains compact below a critical shear

value but shows a globule-stretch transition above this criti-

cal shear value.89 The transition was reported to be more

pronounced in strong extensional ow.

Lemak et al.90 later compared the conformational instabil-

ities of a minimalist b-barrel protein in both uniform andelongational ow elds using MD simulations. It was

reported that whereas unfolding was abrupt in elongational

ow, the uniform ow elds presented a multi-step unfold-

ing process, with several intermediates, depending on the

tethered terminus of the protein. In explaining this observa-

tion, it was proposed that whereas every monomer (i.e.,

amino acid residues) experienced a destabilizing hydrody-

namic drag in elongational ow, only the terminal monomer

experiences this force in uniform ow. Hence, in uniform

ow, deformation of the protein follows an unzipping mech-

anism where the b-strands are unfolded one at a time, lead-ing to the evolution of intermediate states. More importantly,

it was evident that the nature of the applied external force

dictated the preferred mechanism for protein unfolding.

Szymczak and others observed similar ow effects on

employing Brownian dynamics simulations to examine ow-

induced stretching of integrin and ubiquitin.91,92 They noted

that the hydrodynamic drag on a grafted protein chain in

extensional ow increases along the chain length as the poly-

mer unfolds. Therefore, the initiation of unfolding triggers a

positive feedback mechanism which leads to the rapid unravel-

ing of the entire chain to the exclusion of intermediate states.

Indeed, it had previously been predicted that the uid drag

acting on an initially coiled polymer increases as the molecule

unfolds due to the decreased hydrodynamic interactions

between its constituent monomers.84,93 In addition, the

authors observed that whereas the grafted protein chains

unfold through a series of intermediate states in uniform ow,

consistent with that reported earlier,90 synthetic helices unrav-

eled smoothly with no detectable intermediate states.91 This

highlights the fact that proteins and homopolymers do indeed

differ in their hydrodynamic properties in shear ow. Further-

more, it was also noted that the array of metastable conforma-

tions observed during protein unfolding in uniform ow was

absent in mechanical pulling measurements in a force clamp

apparatus such as the atomic force microscope.

FIGURE 4 Change in the helix content of PLL as a function of

shear strain. The gure shows that the a-helix-PLL structure (68.3kDa) is stable below a strain value of 105. For clarity, the shear rates

plotted are: (&) 74 s21, (^) 302 s21, (*) 518 s21, and (l) 715s21. For clarity, error bars representing 6 standard deviation areshown only for the sample sheared at 518 s21.55

Effects of Shear Flow on Protein Structure and Function 741

Biopolymers

A subsequent study examined the inuence of hydrody-

namic interactions, which facilitate the folding of protein

molecules into compact native structures, on the mechani-

cal stretching of proteins using ubiquitin as a model sys-

tem.94 Mechanical stretching at constant speed, at constant

force, and through uid ow was examined. It was found

that hydrodynamic interactions (cohesive forces) between

amino acid residues facilitated unfolding (stretching)

when a constant speed or force was applied to grafted

ubiquitin. This observation was explained on the basis

that an amino acid residue, which is pulled away from

the bulk protein, creates a ow which drags other residues

with it. In contrast, these cohesive forces were found to

counter uid forces by screening monomers concealed in

the protein matrix from the drag in the ow eld, thereby

hindering ubiquitin unfolding in both uniform and elon-

gational ow elds.94 It has been reported that hydrody-

namic interactions increase with increasing protein size

and hence inuence the critical shear rate required to

unfold a protein of radius R.88 However, the authors note

that this prediction pertains to solutions with high solvent

viscosity and low interfacial tension.

Recently, Sulkowska and Cieplak applied the coarse-

grained MD model to investigate the resistance of 7510 pro-

teins to mechanical stretching at a constant speed.95 Pulling

was done by the termini to determine the maximum force

of resistance, Fmax, of the protein chains. They restricted

their study to non-fragmented protein chains deposited in

the Protein Data Bank by August, 2005 and between 40 and

150 amino acid residues long. For specic sequential lengths

of proteins, the average Fmax increased with increasing chain

length. That is, longer protein chains showed better resist-

ance relative to shorter chains. In relation to conforma-

tional classes, a-proteins were found to be weak and gener-ated small Fmax values relative to b- and ab-proteins. Specif-ically, immunoglobulins and transport protein homologies

were shown to yield larger Fmax values. However, the

authors acknowledged that proteins with multi-a-domainsmay show a signicantly high resistance to mechanical

stretching. In total, 134 proteins were considered to be

strong with a threshold force of 170 pN. The authors pro-

posed that the mechanical strength of the strongest proteins

arises from a clamp consisting of long parallel b-strandswhich are stabilized by a network of intra- and intermolecu-

lar interactions.

Implications in Physiology and Bioprocessing

Proteins are known to show a strong conformation-function

relationship. Destabilization of the native protein structure

can potentially lead to the evolution of dysfunctional, aggre-

gation-prone species with undesirable effects.4 Reports in the

literature demonstrate that shear, which is a common stress

in both physiology20,58,96 and bioprocessing,13,18 can induce

protein deformation.97 Therefore, it is important to analyze

the relevance of shear stress to protein molecules under phys-

iological ow and in bioprocessing.

Pulsatile blood ow generates non-uniform shear stress

throughout the arterial system during circulation.96 Arterial

shear rates peak at 1640 s21, 96 reaching 104 s21, 58 in partly

clogged coronary arterioles. The literature contains a signi-

cant number of reports98107 (and references therein) linking

the haemodynamic stress generated on arterial walls during

microcirculation, specically in regions of relatively low

shear stress, to the pathogenesis of atherosclerosis through

the modulation of endothelial function.108 A characteristic

hallmark of this disease state is the deposition of amyloid-

like protein aggregates as plaque on arterial walls. Arterial

amyloid-plaques may trigger vascular inammation, weaken

the elasticity of blood vessels, and alter biochemical reactions

such as lipid metabolism.109 Interestingly, it has been shown

that molecular connement enhances the deformation of

entangled polymers under squeeze ow,110 a condition which

models pulsatile blood ow. Similarly, there are a consider-

ably number of reports27,34,35,42,59,60,111113 citing haemody-

namic shear stress as the primary facilitator of the proteolytic

cleavage of large vWF multimers freshly released from endo-

thelial cells as well as vWF-mediated platelet adhesion during

haemostasis. The accumulation of large vWF multimers in

the plasma may lead to disease states including thrombotic

thrombocytopenic purpura, microvascular occlusion and

atherosclerosis, due to the development of microvascular

thrombi resulting from the complexation of the hyperactive

vWF multimers and platelets at regions of low physiological

shear.63,114 On the other hand, the smaller size of circulating

vWF multimers, resulting from proteolysis, enhance their

haemostatic potential in high shear regimes as occurs during

arteriolar bleeding.62 The inuence of shear stress on the

activity of platelets and vWF has been discussed in other

review articles.57,58,63,115

Others have linked the deterioration in cerebral microcir-

culation (cerebral hypoperfusion) to the onset of protein

conformational disorders such as Alzheimers disease.116118

It is suggested that reduced cerebral blood ow may replace

old age as a risk factor for Alzheimers disease.118 It also pro-

posed that haemodynamic shear stress regulates insulin-like

growth factor-I activity indirectly, and possibly its effect on

vascular pathologies.119

It is noteworthy that although protein conformational

diseases and vascular disorders have received considerable

attention in research, the role of physiological shear stress in

742 Bekard et al.

Biopolymers

the initiation and/or progression of these debilitating diseases

remain to be fully understood.

In the case of bioprocessing, shear stresses are present dur-

ing the separation, purication, shipping, and handling of

protein based products.13 For example, processing steps

involving ultraltration reactors,120,121 stirred tanks,122,123

homogenizers,124 and pumps53,125 induce high shear stresses.

Simulated lobe pump studies have shown that high shear

stress in these processing steps is sufcient to cause minor

changes to protein structure.125 These structural changes

may trigger the exposure of hitherto buried hydrophobic

regions to the aqueous environment, initiating intra/inter-

molecular hydrophobic interactions leading to aggregation.

The simulation further suggests that shear stresses may have

played a crucial role in the protein aggregation behavior

observed in a previous lobe pump study.126 Indeed, protein

aggregation and particle formation during processing can

affect the efciency of protein recovery, efcacy, safety, and

product shelf-life.11 Furthermore, therapeutic protein aggre-

gates are associated with a range of clinical side effects

including non-specic anti-complementary activity leading

to anaphylactic shock.127,128 In addition, insoluble aggregates

can cause immune responses, lodge in the lung capillary bed,

and block blood vessels.129131

The design of experiments to establish the effect of uid

shear stress alone on protein structure and function has

proved difcult. Existing shear devices generate other stresses

including surface interactions and enhanced proteinair con-

tacts, which complicates data interpretation.121,132,133 How-

ever, it has been established that the shear-stability of a pro-

tein molecule depends on: (i) its primary structure and mo-

lecular weight (ii) the magnitude of shear strain and the

duration of its application, and (iii) the viscosity of its sur-

rounding medium.31,64,65 Therefore, these factors must be

taken in consideration for a complete understanding of the

shear-stability of protein systems. Advancing knowledge in

this eld will be crucial for the design of processes, especially

in the bioprocessing industry, to ensure protein stability,

maintain functionality, and promote process yield.

Future development of protein based products will

increasingly require several areas of improvement, especially

in-process-monitoring of the stability of protein solutions.

This will require the development of experimental techniques

to allow the measurement of shear stress on proteins of inter-

est. This is imperative as several studies show that individual

protein systems demonstrate unique ow properties in a

given ow eld. Additionally, it will be essential to appreciate

the shear ( _ct) thresholds beyond which particular proteinsystems destabilize in solution. This will inform the develop-

ment of new processing equipment to improve efciency

during production. It is noteworthy that knowledge from

such studies could contribute to our understanding of the

purported shear-induced pathogenesis of vascular and pro-

tein conformational disorders in vivo.

CONCLUSIONProtein unfolding and aggregation is an issue of concern

both in vitro and in vivo, specically in bioprocessing and in

the pathogenesis of disease. Shear rates less than 103 s21 have

been found to signicantly alter the three-dimensional struc-

ture of globular proteins, leading to aggregation and amyloid

bril formation. The strain history ( _ct) is found to be an im-portant parameter in the shear-effect. Therefore, even in

environments where ostensibly low shear is generated can

lead to shear-induced protein structural change. Given the

diversity of protein species, and the diversity of molecular

sizes, conformations and nature of intramolecular interac-

tions, the susceptibility of proteins to shear can vary. It is

clear that high shear generating processes should be avoided

in bioprocessing. Shear also exists in the circulatory system

and could be a signicant contributor to the pathogenesis of

certain diseases by facilitating the aggregation of aberrantly

folded proteins.

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