Evidence For Metastable States Of Lysozyme Revealed By High Pressure FTIR Spectroscopy

Abstract

Metastable protein conformations play an important role in the folding process because such partially disordered states can be gates for the misfolding pathway, leading sometimes to pathological structures, like fibrous aggregates. High pressure is a very useful tool in the study of metastable states, since application of the pressure is fully reversible, contrary to chemical agents. FTIR spectroscopy allows us to follow simultaneously the secondary structure, the packing (tertiary structure) and the aggregation of the protein using the amide I, amide II and the 1616 cm-1 bands respectively. We performed a systematic study on the temperature-pressure phase diagram of lysozyme and found a two-step unfolding profile both in the pressure and temperature directions. Hydrogen/deuterium exchange results show evidence for the molten globule formation at 57°C @130 MPa and 580 MPa @30°C, which are considerable lower values than those of the complete unfolding (e.g. 75°C@130MPa).Refolding of the protein after pressure unfolding is a slow process, with a time constant in the range of hours. Partially refolded structures present in this time range have different aggregation propensity. The kinetics of the aggregation has a biexponential character with a time constants of 1060 and 8600 s at 40C ambient pressure. Analyzing the time dependence of the amide I band shape we found that the strengthening of the intermolecular hydrogen bond network was accompanied by decrease of the folded secondary structure content.Moderate pressure of 300 MPa was found to be able to dissociate the aggregates, while the secondary structure is not yet unfolded. This fact together with the small temperature effect on the aggregation kinetics suggests that the rate of the aggregation of the metastable conformations is determined by the high activation volume rather than the high activation energy.