Pressure/temperature effects on protein flexibilty from acrylamide quenching of protein phosphorescence.

Pressure is an effective modulator of protein structure and biological function. The influence of hydrostatic pressure (</=3 kbar, 10-50 degrees C) on conformational dynamics was assessed from the rate of migration of acrylamide through the protein interior. Migration rates in apoazurin, alcohol dehydrogenase and alkaline phosphatase were obtained from the phosphorescence quenching rate constant (kq) of the deeply buried Trp residues. The dominant effect of applied pressure is to slow the diffusion process, although at low temperature, high pressure may also accelerate it. For apoazurin, alcohol dehydrogenase and alkaline phosphatase the activation free volumes, DeltaV(obs), derived from the pressure-dependence of kq, ranges from +10, +16 and +20 ml mol(-1)at 50 degrees C to -20, +5 and 0 ml mol(-1)at 10 degrees C, respectively. Analysing DeltaV(obs) in terms of a positive contribution from cavity expansion and a negative one from peptide hydration, the results emphasise that whereas at warm temperature the formation of cavities plays a dominant role in the migration process, at low temperature the required flexibility may be conferred by internal protein hydration. The relatively small magnitude of both DeltaV(obs) and the activation enthalpy (DeltaH=10-20 kcal mol(-1)) indicates that acrylamide diffusion jumps inside these proteins appear to involve relatively small amplitude structural fluctuations not requiring major unfolding-like transitions. The implication of these findings for the thermodynamic stability of proteins under pressure is discussed.