Multi-step thermally induced transitions of β-lactoglobulin – An in situ spectroscopy approach

An in-situ approach based in multiple spectroscopic techniques and benchmarked with DSC was used to characterise β-Lg thermally-induced transitions. The methodology applied overcomes previously reported limitations by ensuring similar experimental conditions in different determinations, non-aggregation conditions and allowing differentiation between fluorescent variations due to collisional quenching and structural modifications. These experimental improvements along with the correlation of complementary data from the assessment of several unfolding-related events, allowed a real time, precise and detailed description of the unfolding/refolding pathways of β-Lg. The existence of a complex multi-step unfolding mechanism was confirmed, with a focus on the reversible conformational changes. The elusive unfolding intermediates were characterised in terms of structural swelling, hydrophobic sites accessibility and tryptophan exposure. This approach allowed establishing a clear order of events during thermally-induced structural changes, representing a step forward in the understanding of protein stability and interactions, useful, e.g., when establishing heat treatments of dairy products.