Résumé
High pressure, combined with high temperature, is an interesting tool to explore the energy landscape of protein unfolding and aggregation. This approach can be employed for the study of many proteins, simply by following pressure dependent changes in their spectral properties (UV absorbance in the 4th derivative mode, intrinsic fluorescence, and fluorescence in the presence of extrinsic probes). The kinetics of these conformational changes can be studied by the pressure jump induced relaxation. This method is illustrated with a model protein, ribonuclease A and several of its mutants. Our results indicate complex and non-conventional pathways for protein unfolding. We have then applied this concept to study proteins of interest in neuro-degenerative diseases (ataxin-3, and prion proteins). In both cases, unfolding occurs in at least 2 pressure dependent steps. At elevated pressure, an amyloid precursor appears to be formed. Furthermore, by applying a moderate pressure, heat induced prion protein unfolding can be studied in the absence of protein aggregation.