Shear Pulses Nucleate Fibril Aggregation

Abstract

We have studied the effect of shear flow on the formation of amyloid fibrils of the whey protein blactoglobulin. b-Lactoglobulin aggregates into long, thin, and semiflexible fibrils upon heating at low pH and low ionic strength. Solutions with a protein concentration of 0.5% (w/w) were used, and the formation of fibrils was quantified with flow-induced birefringence, a proportional measure of the length concentration of the fibrils. From the decay of the birefringence after cessation of the flow, a length distribution could be fitted. Pulsed and continuous shear treatment of the samples resulted in a comparable enhancement of the fibrillar growth as compared to the fibrillar growth under quiescent conditions. This indicates that the onset of shear flow is the key parameter for the enhancement of fibrillar growth and not the continuous shear flow itself. This behavior is comparable to a nucleation-like process, during which preaggregates of the fibrils are induced during the onset of the flow and orthokinetic coagulation is absent. However, a difference was present in the length distribution between the pulsed and continuously sheared samples, which can be explained by the homogenizing effect of shear flow.

The effects of shear flow on protein structure and function

“…These observations lead to the conclusion that brief mechanical perturbation of an aggregation prone protein solution was enough to enhance fibril formation. 77 That is, shear flow only triggered the nucleation of fibril formation but did not influence the polymerization of preaggregates into mature fibrils, discounting orthokinetic coagulation under the experimental conditions. However, the fibrils formed from the continuous shear treatment showed a smaller variance in Gaussian length distribution relative to those from the pulse shear treatments, indicating a homogenizing effect of the continuous shear treatment.…”

“…For example, a shear-induced nucleation, without flow enhanced aggregation, of preheated b-lactoglobulin solutions (0.5 wt%) at low pH has been reported. 77 b-lactoglobulin solutions were exposed to Couette flow in a rheometer, at a moderate shear rate of 200 s 21 over 5 h, while measuring the flow-induced birefringence. Thermal treatment of the b-lactoglobulin samples triggered the formation of metastable pre-fibrillar aggregates, without which fibril formation was absent in the sheared samples.…”

“…25 In addition, if shearing was performed prior to thermal treatment, a rapid enhancement of fibrillar species still occurred, suggesting a shear-induced formation of the precursor nuclei required for fibril development as previously reported. 77 Interestingly, at shear rates [500 s 21 , fibril degradation, possibly arising from the persistent stretching (extensional flow) events in the flow field, was observed. A further study 78 revealed that a sufficiently high b-lactoglobulin concentration ([ 3 wt%) was required to initiate fibril formation when samples were heated and sheared simultaneously.…”

Enzyme-Induced Formation of β-Lactoglobulin Fibrils by AspN Endoproteinase

“…Previous work showed already that fibrils of β-lactoglobulin could be formed at room temperature after a short heat treatment of 2 h at 90°C, during which no fibril formation took place yet. 12 At pH 8, the fluorescent intensity increased in time, while random aggregates were formed. At pH 2, random aggregates (smaller in size than at pH 8) and fibrils were observed, which led to a higher fluorescent intensity than at pH 8.…”

FTIR Spectroscopic Study of Heat-Induced β-Lactoglobulin Solution under Flow Field

“…The nucleation growth mechanism of fibrillogenesis can be influenced by several factors such as seeding [20], stirring [21], shearing [22] and sonication [23]. Various techniques have been used earlier to analyze the effect of flow field on the fibrillogenesis of β-lactoglobulin and WPI (whey protein isolate) [24][25][26]. We have analyzed the effect of flow field (stirring) on the fibrillogenesis of β-lactoglobulin at pH 2 [27] and also pH 7 with and without glucose [28] incubated at ≥ 80°C for 24 h. The present work is a continuation of our previous work in which we are characterizing the secondary structure of β-lactoglobulin fibrils by using Fourier transform infrared (FTIR) spectroscopy.…”