PRESSURE EFFECTS ON PROTEIN FLEXIBILITY MONOMERIC PROTEINS

Alterations in flexibility of monomeric proteins induced by hydrostatic pressure in the predenaturational range (less than or equal to 3 kbar) were probed through the decay kinetics of tryptophan phosphorescence. With apoazurin, ribonuclease T-1, wild-type and VB7G mutant and phosphoglycerate kinase, pressure effects on the triplet lifetime (tau) and the amplitudes of multicomponent decays emphasize that subtle changes in conformation are ubiquitous. With apoazurin the increase in tau attests to a tightening of the protein core that is enhanced at high temperature. On the contrary, tau decreases with ribonuclease T-l, wild-type and mutant, and with phosphoglycerate kinase, indicating that pressure induces a greater flexibility to protein regions in proximity to the surface of the macromolecule. For phosphoglycerate kinase the decrease in tau and the parallel increase in fluorescence intensity and red-shift of the fluorescence spectrum unveil an ''unfolding'' like transition with midpoint pressures of 1 1 kbar at 5 degrees C and 1 B kbar at 25 degrees C. Evidence that unfolding of the C-domain of this protein is, however, less than complete is provided by a Delta G(0) that is about half of that obtained by denaturation in guanidine hydrochloride and also by the ability of this structure to undergo conformational drift.