期刊
FOOD HYDROCOLLOIDS
卷 112, 期 -, 页码 -出版社
ELSEVIER SCI LTD
DOI: 10.1016/j.foodhyd.2020.106174
关键词
beta-lactoglobulin; Amyloid aggregates; Site-directed spin labeling; EPR; Simulation
资金
- German Research Foundation (DFG) [SPP 1934 DiSPBiotech, 273937032]
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.
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.
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