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.

中文翻译：

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自旋标记的β-乳球蛋白第二部分的淀粉样蛋白聚集：淀粉样蛋白聚集后自旋标记的蛋白质和肽序列的鉴定

摘要 建立了天然β-乳球蛋白（β-lg）的定点自旋标记（SDSL）。结合电子顺磁共振 (EPR) 和胰蛋白酶消化后的质谱分析表明，自旋标记位点特异性结合，但并非针对所有五个半胱氨酸残基达到各种优选和可重复的程度。MTSSL 和碘乙酰氨基-代理自旋标记 (IPSL) 分别与 H 链有 80% 和 60% 的可靠结合，并结合在一个光谱成分中并埋在蛋白质核心中。在 pH 2 下加热孵育和分级后，所有标记的侧链（肽）都是淀粉样蛋白和非淀粉样蛋白部分的一部分，即使它们无法检测到淀粉样蛋白结构。假设来自无规卷曲的带有 Cys160 的肽的 IPSL 标记侧链在非极性环境中被并入小的非淀粉样聚集体中。在 pH 3.5 加热后，假设先前的 α-螺旋的重排在部分展开期间从自主折叠域转移，这提高了 β-折叠对水/DMSO 环境的可及性。β-折叠很可能通过分子间β-折叠的积累而密集堆积，这表明淀粉样结构可以由整个初级β-lg结构的构建块形成。双电子-电子共振 (DEER) 证实，在整个蛋白质聚集体的一维排列中，淀粉样蛋白部分内标记的空间分布类似于一串珍珠。因此，