Secondary structure and folding stability of proteins adsorbed on silica particles – Pressure versus temperature denaturation

Abstract

We present a systematic study of the pressure and temperature dependent unfolding behavior of proteins that are adsorbed on silica particles. Hen egg white lysozyme and bovine ribonuclease A (RNase) were used as model proteins, and their secondary structures were resolved by Fourier transform infrared (FTIR) spectroscopy in the temperature range of 10–90°C and the pressure range of 1–16,000bar. Apparently, the secondary structures of both proteins do not change significantly when they are adsorbing on the silica particles. Remarkably, the changes of the secondary structure elements upon protein unfolding are very similar in the adsorbed and the free states. This similarity could be observed for both lysozyme and RNase using both high pressures and high temperatures as denaturing conditions. However, the pressures and temperatures of unfolding of lysozyme and RNase are drastically lowered upon adsorption indicating lower folding stabilities of the proteins on the silica particles. Moreover, the temperature ranges, where changes in secondary structure occur, are broadened due to adsorption, which is related to smaller enthalpy changes of unfolding. For both proteins, free or adsorbed, pressure-induced unfolding always leads to less pronounced changes in secondary structure than temperature-induced unfolding. In the case of lysozyme, high pressure also favors a different unfolded conformation than high temperature. Overall, the results of this study reveal that adsorption of proteins on silica particles decreases the folding stability against high pressures and temperatures, whereas the unfolding pathways are mainly preserved in the adsorbed state.