Amyloid aggregation of spin-labeled β-lactoglobulin. Part I: Influence of spin labeling on amyloid aggregation

Abstract

Site-directed spin labeling (SDSL) of the natural food protein β-lactoglobulin (β-lg) was established with the aim of characterizing amyloid aggregation while explicitly avoiding the usual manipulation of primary protein structure. For its successful application, spin labels must not alter secondary protein structure or the formation of β-lg amyloid aggregates. The two spin labels—the MTSSL (flexible S–S binding) and Iodoacetamido-proxyl spin label (IPSL) (more rigid C–S binding)—were used for amyloid aggregation at pH 2 and pH 3.5. At pH 3.5, IPSL caused minor changes in the secondary protein structure, where it reduced intra- and intermolecular β-sheets as determined by ATR-FTIR. Analysis of the extent of amyloid aggregation using thioflavin T fluorescence indicated that the spin probes interfered with the binding of the fluorescent probes to the β-sheets. Non-amyloid and amyloid fractions were obtained from the amyloid aggregated system by ultrafiltration (300 kDa), which also proved to be equivalent and independent of the spin labeling process. Atomic force microscopy and size-exclusion chromatography results suggest the same building blocks and morphologies between unlabeled and spin-labeled proteins; therefore, substantial changes in the behavior of β-lg during amyloid aggregation can be excluded. Ultimately, 10–17% of the β-lg molecules were labeled so that the SDSL approach can be used to label the natural food protein at these low concentrations without affecting protein conformation or the formation of amyloid aggregates, which may subsequently provide deep insights into the aggregation mechanism of food proteins under processing conditions.