High-pressure effects on protein phenomena: Crystallization, aggregation and folding

Several different protein association phenomena (crystallization, aggregation, oligomer association and the dissolution and refolding of aggregates) were investigated at high pressure. Equipment and techniques were developed for collecting in situ high-pressure measurements. Volume changes of the association processes were determined from measurements taken before and after pressurization as well as in situ and used to interpret the nature of the effects and develop mechanisms for the observations. The crystallization of subtilisin is arrested at high pressure and the extent of arrest is largely explained by the increase in solubility with pressure. The aggregation of recombinant human interferon-gamma (rhIFN-gamma) is dissociation-dependent and well characterized by a modified Lumry-Eyring model. The perturbation that is required for the formation of the transition-state species leading to aggregate competent monomer is small in comparison to the difference between native and dissociated states. Pressure is effective in rapidly dissolving and refolding rhIFN-gamma in the absence of chaotropes and at concentrations that are high in comparison to traditional refolding protocols. Pressure is a valuable tool for investigating protein phenomena, elucidating molecular-level information on the processes and has potential application for processing proteins at the production scale.