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Formation of β-Lactoglobulin Aggregates from Quite, Unfolded Conformations upon Heat Activation

Peixoto, Paulo D. S., Trivelli, Xavier, André, Christophe, Moreau, Anne, Delaplace, Guillaume
Langmuir 2018 v.35 no.2 pp. 446-452
ambient temperature, beta-lactoglobulin, calcium, heat, ions, molecular conformation, nuclear magnetic resonance spectroscopy, pH, protein unfolding
In presence of calcium ions, β-lactoglobulin (BLG) unfolds and subsequently aggregates after heating. This process has important pharmaceutical and agroalimentary applications. Nowadays, the molecular mechanism of unfolding and BLG aggregation, and the role of calcium in the mechanism, is poorly understood. Actually, in most studies, data have been acquired at room temperature, after heating and after aggregation, which makes it difficult to establish a clear causal–temporal relation between calcium binding, heat, and aggregation. Thus, the goal of the present study is to get accurate, nanoscale data about the molecular events leading to BLG unfolding and calcium-dependent aggregation. The molecular transformation of BLG during heating has been investigated, using the NMR pulse field gradient technique, operating in a high field (900 MHz). Thanks to this technique, the molecular conformation of newly formed unfolded BLG molecules can be distinguished in a large pool of native ones. The present work shows that BLG at neutral pH at 65 °C displays fast, cooperative-like unfolding, in which no long-lived intermediary state (as a molten globule one) is detected, before aggregation. These data also indicate that calcium ions bind unfolded BLG in specific sites which might be a necessary feature to form the aggregate. Finally, these data also provide an NMR-based methodology to monitor the rate of protein unfolding using NMR.