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

Jacqueline Lux*, Mykhailo Azarkh, Laura Fitzner, Julia K. Keppler, Karin Schwarz, Malte Drescher, Anja Steffen-Heins

*Corresponding author for this work

Research output: Contribution to journalArticleAcademicpeer-review

Abstract

Site-directed spin labeling (SDSL) of natural β-lactoglobulin (β-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 Cys160 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 α-helix was assumed to shift from the autonomous folding domain during partial unfolding, which improved the accessibility of β-sheets to the water/DMSO-environment. β-sheets were likely densely packed by the accumulation of intermolecular β-sheets, which suggests that amyloid-like structures can be formed from building blocks of the entire primary β-lg structure. Double electron-electron resonance (DEER) confirmed that the spatial distribution of labels within the amyloid-like fraction in a one-dimensional 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.

Original languageEnglish
Article number106174
JournalFood Hydrocolloids
Volume112
DOIs
Publication statusPublished - Mar 2021

Keywords

  • Amyloid aggregates
  • EPR
  • Simulation
  • Site-directed spin labeling
  • β-lactoglobulin

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