Amyloid aggregation of spin-labeled β-lactoglobulin. Part II: Identification of spin-labeled protein and peptide sequences after amyloid aggregation

Site-directed spin labeling (SDSL) of natural beta-lactoglobulin (beta-lg) was established. Combined electron paramagnetic resonance (EPR) and mass spectrometric analysis following tryptic digestion demonstrated that spin labels bind site-specifically but are not directed to all five cysteine residues to various preferred and reproducible extents. MTSSL and iodoacetamido-proxyl spin label (IPSL) were 80 and 60% reliably bound to the H strand, respectively, and combined in one spectral component and buried in the protein core. After heat incubation at pH 2 and fractionation, all labeled side chains (peptides) were part of the amyloid and non-amyloid fractions, even if they could not detect amyloid structures. It was assumed that the IPSL-labeled side chains of peptides with Cys(160) from random coil were incorporated into small non-amyloid aggregates in non-polar environments. After heating at pH 3.5, a rearrangement of the previous alpha-helix was assumed to shift from the autonomous folding domain during partial unfolding, which improved the accessibility of beta-sheets to the water/DMSO-environment. beta-sheets were likely densely packed by the accumulation of intermolecular beta-sheets, which suggests that amyloid-like structures can be formed from building blocks of the entire primary beta-lg structure. Double electron-electron resonance (DEER) confirmed that the spatial distribution of labels within the amyloid-like fraction in a onedimensional arrangement of the entire protein aggregates was similar to a string of pearls. Thus, SDSL of proteins containing several cysteine residues can be used to gain deep insights into the aggregation mechanism of proteins under food processing conditions.

Automated summary

SDSL of natural beta-lactoglobulin revealed that different spin labels bind to specific sites to varying extents, affecting the formation of amyloid-like structures. The spatial distribution of labels within protein aggregates was confirmed to be similar to a string of pearls, indicating a potential mechanism for amyloid formation.