Proteins under pressure.

T oday, 3D models of proteins are often constructed entirely by a computer. Building old-fashioned wire models may have been time-consuming, but it was not drudgery. To the contrary, it was a labor of love. One could not fail to marvel at the way in which the successive amino acids fitted together, with all the interactions making physical sense. However, the packing was not perfect. Here and there, small unfilled spaces remained, and as shown by Roche et al. (1) in PNAS, these cavities come at a price, particularly with regard to pressure denaturation. Given the current interest in exploring the deepest regions of the ocean, it might be noted that the pressure at the bottom of the Mariana Trench is about 1,100 bar (∼1,100 atm). Early studies showed that a number of proteins remain folded at substantially higher pressures. Therefore, hydrostatic pressure, of itself, is not expected to preclude life in the deep. The present experiments (1) use mutants of the small monomeric protein staphylococcal nuclease (SNase). These proteins do not unfold within the working range of the instrument (up to 3,000 bar), but unfolding transitions can be monitored by adding supplementary denaturant (0.8–1.5 M GuHCl). Clearly, SNase would be perfectly stable under ambient conditions in the Mariana Trench. It is well known that the creation of an artificial cavity in the core of a folded protein (e.g., by replacing a large nonpolar residue with a small one) is destabilizing. The larger the volume of the created cavity, the greater is the loss in protein stability (2). Although such cavities destabilize proteins against heat and denaturants, the mechanism by which pressure unfolds proteins has been somewhat contentious. An especially informative experiment was carried out by Akasaka and coworkers (3) using a subdomain of the c-Myb transcription factor. This folded 52residue domain has a naturally occurring cavity with a volume of 33.1 Å. Using pressure alone, the domain becomes almost entirely unfolded at 3,700 bar. There is a mutation, valine 103 to leucine (V103L), which fills the naturally occurring cavity and also greatly increases the resistance of the protein to pressure denaturation. At 3,700 bar, the mutant has only begun to unfold and appears to require pressure in excess of 8,000 bar to denature completely. The apparent reduction in volume associated with unfolding of the native (cavity-containing) domain (ΔΔVu = 35.3 Å) agrees well with the volume of the cavity, suggesting that it is the collapse of the cavity that is responsible for protein unfolding.