Analysis of cold denaturation mechanism of β-lactoglobulin and comparison with thermal denaturation from Raman spectroscopy investigations

摘要

Cold- and heat-induced β-lactoglobulin (BLG) transformations have been analyzed in the presence of 4 M urea, from Raman spectroscopy investigations carried out simultaneously in the low wavenumber range (10-400 cm ~(-1)) and in the amide I region (1500-1800 cm ~(-1)). These investigations show common features between the denaturation processes at low and high temperatures. The denatured states are reached via an intermediate state characterized by a soft tertiary structure without detectable conformational changes. This intermediate is intimately connected with a tetrahedral hydrogen-bond structure of water which extends over a limited range. It is shown that the disruption of the hydrogen-bond network of D _2O has an important consequence on the solvent dynamics, which controls protein dynamics and is characterized by an anharmonic behavior. By monitoring the amide I mode, conformational changes are detected at low temperature (below 5 °C) and determined to be similar to those detected at high temperature in the presence of urea near 65 °C, and in the absence of urea near 80 °C. The conformational changes are described as a loss of α-helix structures and a concomitant formation of β-sheets. The temperature dependence of the amide I wavenumber in BLG dissolved in the 4 M urea aqueous solution was interpreted on the basis of a two-state model, leading to the protein stability curve related to its molecular conformation.