Site-directed spin labeling for the evaluation of amyloid and non-amyloid aggregation mechanisms of β-lactoglobulin using electron paramagnetic resonance spectroscopy and mass spectrometry
The approach of site-directed spin labeling can be implemented for the food protein β lactoglobulin (β lg), by adjusting the pH value and the spin label/β lg ratio, although it contains five cysteine residues, and used to characterize the amyloid aggregation mechanism of β lg. Further, the surface activity of the amyloid aggregate system is determined by the smaller aggregates, peptides, and monomeric sized protein of the non-amyloid fraction due to faster migration to the interface and a higher adsorption rate. The results of this work are mainly based on measurements using electron spin resonance spectroscopy and mass spectrometry. MTSSL and IPSL bind covalently and reproducibly to all five cysteine residues with different preferences. The best labelling conditions for MTSSL are pH 7.5 and an equimolar MTSSL/β-lg ratio. Whereby MTSSL dimers are already formed at a pH value of 7.5. The bonding of the spin labels to β-lg is temperature (90°C) and acid (pH 2) resistant, whereas it is assumed the biradicals are reduced to monoradical dimers during heat incubation. Spin labeling with IPSL reduces the intra- and intermolecular β-sheets at pH 3.5. However, SDSL does not change the morphology and building blocks of amyloid aggregates but interferes with the thioflavin-T measurement. Impact of the label on the secondary protein structure is lower for MTSSL compared to IPSL because of its flexible bond. MTSSL is most sensitive to changes in the arrangement of β-sheets, whereas IPSL is most sensitive to changes in random coil at position Cys160. The amyloid aggregates formed at pH 3.5 could be characterized using SDSL. The monomer-like β-lg aggregates had the form of a pearl chain. However, the spin labeled side chains were not incorporated into amyloid structures formed at pH 2. Fibrils and non-amyloid material act synergistically at the air-water interface because non-amyloid material is embedded into fibrillar network. But the adsorption behavior of the amyloid aggregate system is dominated by the non-amyloid fraction. Only small amounts of amyloid aggregates were necessary to stabilize foams. The airflow used for foaming resulting in gas bubbles caused sufficient shearing to partly degrade fibrils.