Information on changes in heat capacity (¿Cp) of proteins upon unfolding is used frequently in literature to understand possible follow-up reactions of protein denaturation, like their aggregation propensity. This thermodynamic property is intrinsic to the protein's architecture and unfolding and should be independent of the approach used to evaluate it. However, for many proteins, the reported values for ¿Cp vary considerably. To identify whether the origin of these discrepancies lies within the experimental approach chosen and/or in the too simplified unfolding models used in the analysis of the data, we choose ß-lactoglobulin A, a relatively small protein, but disputed for its two-state unfolding, and established its ¿Cp from tryptophan fluorescence, near-UV circular dichroism and differential scanning calorimetric measurements. In view of the large variation for the obtained ¿Cp (between 3.2 and 10.1 ± 0.8 kJ/(mol K)), it is evident that: (1) the sensitivity of different approaches to the structural changes; (2) irreversibility of unfolding; (3) non-ideal two-state unfolding behaviour need to be considered prior to interpretation. While the first two points can be addressed by using multiple approaches, the applicability of the selected unfolding behaviour for the analysis is often less easy to establish. In this work, we illustrate that by checking the wavelength-dependence used to detect protein conformational changes a tool is provided that gives a direct insight in the validity of the interpretation in these studies. An experimentally validated determination of ¿Cp allows a more proper use for the mechanistic understanding of protein denaturation and its follow-up reactions, avoiding pitfalls in the interpretation.
|Journal||International Journal of Biological Macromolecules|
|Publication status||Published - 2005|
- monitor unfolding transitions
- fluorescence methods
- protein stability
- cold denaturation