Formation of β-Lactoglobulin Nanofibrils by Microwave Heating Gives a Peptide Composition Different from Conventional Heating

Abstract

A novel procedure involving microwave heating (MH) at 80 °C can be used to induce self-assembly of β-lactoglobulin (β-lg) into amyloid-like nanofibrils at low pH. We examined the self-assembly induced by MH, and evaluated structural and compositional differences between MH fibrils and those formed by conventional heating (CH). MH significantly accelerated the self-assembly of β-lg, resulting in fully developed fibrils in ≤2 h. However, longer MH caused irreversible disintegration of fibrils. An increase in the fibril yield was observed during the storage of the 2 h MH sample, which gave a yield similar to that of 16 h CH sample. Fourier transform infrared (FTIR) and circular dichroism (CD) spectra suggested that the fibrils formed by the two methods do not show significant differences in their secondary structure components. However, they exhibited differences in surface hydrophobicity, and mass spectrometry showed that the MH fibrils contained larger peptides than CH fibrils, including intact β-lg monomers, providing evidence for a different composition between the MH and CH fibrils, in spite of no observed differences in their morphology. We suggest MH initially accelerates the self-assembly of β-lg due to its nonthermal effects on unfolding, nucleation, and subsequent stacking of β-sheets, rather than promoting partial hydrolysis. Thus, MH fibrils are composed of larger peptides, and the observed higher surface hydrophobicity for the MH fibrils was attributed to the parts of the larger peptides extending out of the core structure of the fibrils.

Evaluation of protease resistance and toxicity of amyloid-like food fibrils from whey, soy, kidney bean, and egg white

“…Such variation in morphology with protein source and environmental conditions has previously been observed for a variety of proteins (Petkova et al, 2005), including those used in this study (Loveday et al, 2011;Tang & Wang, 2010;. The observed diversity in fibril morphology is likely due to the difference in amino acid composition and the varying amount of each peptide chain that is incorporated into the amyloid backbone, as observed for b-lactoglobulin fibrils (Hettiarachchi, Melton, Gerrard, & Loveday, 2012). WPI fibrils were found to be straight, reaching several micrometres in length and about 10 nm in width.…”

“…4 and 6), although the contribution of non-fibrillar peptide components cannot be ruled out completely. The yield of b-lactoglobulin fibrils formed under similar conditions to WPI fibrils in this work has previously been assessed to be as high as 50-80% (Hettiarachchi et al, 2012;Li et al, 2012). While we acknowledge the role of disease related amyloid fibrils and their resistance to common proteases, we found that fibrils formed from food proteins were not directly toxic to in vitro human cells and may even be used as a source of nutrition.…”

Formation of nano-fibrils from the A, B and C variants of bovine β-lactoglobulin

“…(1), are given in Table 1. The acid hydrolysis of b-Lg during selfassembly occurs by the cleavage of Asp-X or X-Asp peptide bonds in the sequence (Akkermans et al, 2008;Dave et al, 2013;Hettiarachchi et al, 2012) and promotes self-assembly by generating assembly-capable peptides that form the building blocks of fibrils (Dave et al, 2013). The differences in k h for b-Lg A, B and C were not significant (p ¼ 0.47, likelihood ratio test), which indicates that the additional site of acid hydrolysis (Asp64, b-Lg A) did not affect the overall rate of hydrolysis of b-Lg A.…”

“…Although the native structure of the three b-Lg variants is largely similar (Bewley, Qin, Jameson, Sawyer, & Baker, 1997), the amino-acid substitutions influence their heat stability, aggregation propensity and the properties of aggregates formed upon heating at neutral pH (Manderson, Creamer, & Hardman, 1999;Nielsen, Singh, & Latham, 1996;Qin, Bewley, Creamer, Baker, & Jameson, 1999), although differences were not apparent at pH 2 (Le Bon, Durand, & Nicolai, 2002). Previous studies of b-Lg fibril formation have used either whey protein isolate (Bolder, Vasbinder, Sagis, & van der Linden, 2007) or a mixture of b-Lg A and B (Akkermans et al, 2008;Hettiarachchi, Melton, Gerrard, & Loveday, 2012). It is not known, therefore, whether the genetic variation in b-Lg A, B and C will have any effect on their selfassemblies.…”

“…The sites of substitutions in the sequences of b-Lg A, B and C have been found in fibril-forming peptides (Akkermans et al, 2008;Dave et al, 2013;Hettiarachchi et al, 2012). Under the highly acidic conditions of self-assembly, the Asp64 substitution in b-Lg A would provide an additional site for acid hydrolysis that is not present in variants B and C. The substitution of Asp49 and Asp101 in the sequence of chicken lysozyme enhanced fibrillar self-assembly at pH 2, in comparison to equine lysozyme (Gln49) and turkey lysozyme (Gly101), by facilitating acid hydrolysis (Krebs, Morozova-Roche, Daniel, Robinson, & Dobson, 2004;Mishra et al, 2007).…”