The use of pressure-jump relaxation kinetics to study protein folding landscapes

Pressure-jump induced relaxation kinetics can be used to study both protein unfolding and refolding. These processes can be initiated by upward and downward pressure-jumps of amplitudes of a few 10 to 100 MPa, with a dead-time on the order of milliseconds. In many cases, the relaxation times can be easily determined when the pressure cell is connected to a spectroscopic detection device, such as a spectrofluorimeter. Adiabatic heating or cooling can be limited by small pressure-jump amplitudes and a special design of the sample cell. Here, we discuss the application of this method to four proteins: 33-kDa and 23-kDa proteins from photo-system II, a variant of the green fluorescent protein, and a fluorescent variant of ribonuclease A. The thermodynamically predicted equivalency of upward and downward pressure-jump induced protein relaxation kinetics for typical two-state folders was observed for the 33-kDa protein, only. In contrast, the three other proteins showed significantly different kinetics for pressure-jumps in opposite directions. These results cannot be explained by sequential reaction schemes. Instead, they are in line with a more complex free energy landscape involving multiple pathways.