Pressure Effects on Protein Flexibility Monomeric Proteins

Alterations in flexibility of monomeric proteins induced by hydrostatic pressure in the predenaturational range (≤3 kbar) were probed through the decay kinetics of tryptophan phosphorescence. With apoazurin, ribonuclease T1, wild-type and V67G mutant and phosphoglycerate kinase, pressure effects on the triplet lifetime (τ) and the amplitudes of multicomponent decays emphasize that subtle changes in conformation are ubiquitous. With apoazurin the increase in τ attests to a tightening of the protein core that is enhanced at high temperature. On the contrary, τ decreases with ribonuclease T1, 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 τ and the parallel increase in fluorescence intensity and red-shift of the fluorescence spectrum unveil an "unfolding" like transition with midpoint pressures of I·l kbar at 5°C and 1·6 kbar at 25°C. Evidence that unfolding of the C-domain of this protein is, however, less than complete is provided by a ΔG° that is about half of that obtained by denaturation in guanidine hydrochloride and also by the ability of this structure to undergo conformational drift. In 70% glycerol, pressure effects on τ of apoazurin are attenuated while for ribonuclease T1 there is a reversal of the tendency with a pronounced increase in τ. With phosphoglycerate kinase glycerol abolishes entirely the "unfolding" transition and all hysteresis effects. A consistent picture of these findings is provided in terms of the location of the probe and of the opposing effects that pressure exerts on protein flexibility by reducing internal cavities and increasing the hydration of the polypeptide.