β-Lactoglobulin Self-Assembly: Structural Changes in Early Stages and Disulfide Bonding in Fibrils

Abstract

Bovine β-lactoglobulin (β-Lg) self-assembles into long amyloid-like fibrils when heated at 80 °C, pH 2, and low ionic strength (<0.015 mM). Heating β-Lg under fibril-forming conditions shows a lag phase before fibrils start forming. We have investigated the structural characteristics of β-Lg during the lag phase and the composition of β-Lg fibrils after their separation using ultracentrifugation. During the lag phase, the circular dichroism spectra of heated β-Lg showed rapid unfolding, and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of samples showed increasing hydrolysis of β-Lg. The SDS-PAGE profiles of fibrils separated by ultra centrifugation showed that after six hours, the fibrils consisted of a few preferentially accumulated peptides. Two-dimensional SDS-PAGE under reducing and nonreducing conditions showed the presence of disulfide-bonded fragments in the fibrils. The sequences in these peptide bands were characterized by in-gel digestion electrospray ionization (ESI)-MS/MS. The composition of solubilized fibrils was also characterized by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS/MS. Both MS analyses showed that peptides in fibrils were primarily from the N-terminal region, although there was some evidence of peptides from the C-terminal part of the molecule present in the higher molecular weight gel bands. We suggest that although the N-terminal region of β-Lg is almost certainly involved in the formation of the fibrils, other peptide fragments linked through disulfide bonds may also be present in the fibrils during self-assembly.

On the role of peptide hydrolysis for fibrillation kinetics and amyloid fibril morphology

“…17,19,23 Similar regions have also been observed to play important roles to brillation of b-lactoglobulin at pH 7.0, 37 C and 5 M urea. 38 We investigated the composition of the puried WPI brils (72 h brillation time) using MALDI-TOF MS and the same protocol as employed by Akkermans et al 17 We found that the main peaks in the mass spectra correspond to the molecular masses reported for N-terminal segments 1-32/33 (3439/3554 Da) and 12-32/33 (2238/2353 Da) and for the C-terminal segment 138-162 (2917 Da) (Fig.…”

“…16 Fibrillation of WPI at low pH and high temperature has been suggested to proceed through hydrolysis of the proteins into smaller peptide fragments that spontaneously assemble into PNFs. [17][18][19] Although other whey proteins, e.g. a-lactalbumin, can also form amyloid-like brils, 20 PNFs formed under the applied conditions have been shown to be built exclusively from b-lactoglobulin-derived peptides.…”

“…21 The bril assembly has been correlated with the polypeptide hydrolysis reaction 18,22 and mass spectrometry (MS) has been used to identify the peptide building blocks of the brils. 17,19,23 The consensus of these investigations is that the two polypeptide segments, one in the most N-terminal part of b-lactoglobulin (residues 1-53) and one in the C-terminus (residues 138-162), constitute the key building blocks. The details of the bril structure are, however, not known.…”

Role of dietary antioxidant (−)-epicatechin in the development of β-lactoglobulin fibrils

“…It has been shown that peptides, rather than the intact protein, are the fibrils building blocks [9]. The existence of disulfide-bound fragments in BLG fibrils has also been shown [10,11].…”

Design of whey protein nanostructures for incorporation and release of nutraceutical compounds in food

“…Several studies point at formation of such structures, mostly from -Lg (Kavanagh et al, 2000;Bolisetty et al, 2012;Dave et al, 2013) and WPI (Ikeda and Morris, 2002;Loveday et al, 2011;Liu and Zhong, 2013). Heating pure a-La or pure BSA at pH 2 is not enough to induce for-mation of fibrillar structures (Bolder et al, 2006a;Bolder et al, 2006b;Bolder et al, 2007).…”