Β-Lactoglobulin Assembles into Amyloid through Sequential Aggregated Intermediates
We have investigated the aggregation and amyloid fibril formation of bovine β-lactoglobulin variant A, with a focus on the early stages of aggregation. We used noncovalent labeling with thioflavin T and 1-anilino-8-naphthalenesulfonate to follow the conformational changes occurring in β-lactoglobulin during aggregation using time resolved luminescence. 1-Anilino-8-naphthalenesulfonate monitored the involvement of the hydrophobic core/calyx of β-lactoglobulin in the aggregation process. Thioflavin T luminescence monitored the formation of amyloid. The luminescence lifetime distributions of both probes showed changes that could be attributed to conformational changes occurring during and following aggregation. To correlate the luminescence measurements with the degree of aggregation and the morphology of the aggregates, we also measured dynamic light scattering and atomic force microscopy images. We evaluated the relative stability of the intermediates with an assay that is sensitive to aggregation reversibility. Our results suggest that initial aggregation during the first 5 days occurred with partial disruption of the characteristic calyx in β-lactoglobulin. As the globular aggregates grew from days 5 to 16, the calyx was completely disrupted and the globular aggregates became more stable. After this second phase of aggregation, conversion into a fibrillar form occurred, marking the growth phase, and still more changes in the luminescence signals were observed. Based on these observations, we propose a three-step process by which monomer is converted first into weakly associated aggregates, which rearrange into stable aggregates, which eventually convert into protofibrils that elongate in the growth phase.
MSU Digital Commons Citation
Giurleo, Jason T.; He, Xianglan; and Talaga, David, "Β-Lactoglobulin Assembles into Amyloid through Sequential Aggregated Intermediates" (2008). Department of Chemistry and Biochemistry Faculty Scholarship and Creative Works. 451.