PRESSURE EFFECTS ON THE STRUCTURE OF OLIGOMERIC PROTEINS PRIOR TO SUBUNIT DISSOCIATION

In studies of pressure-induced subunit dissociation of protein aggrega tes, now widely used to evaluate the association free energy, entropy and enthalpy of very stable complexes, it is assumed that high pressur e does not influence their structure/thermodynamic parameters and that some peculiarities of these equilibria, such as the decrease in subun it affinity at larger degrees of dissociation (alpha) and hysteresis i n alpha/pressure diagrams are imputable to the slow conformational dri ft of isolated subunits. To test this premise, the conformation of dim eric alcohol dehydrogenase from horse liver and alkaline phosphatase f rom Escherichia coli was monitored as a function of pressure (up to 3 kbar) and temperature (0 to 50 degrees C) by means of the intrinsic Tr p fluorescence and phosphorescence emission and binding of the 1-anili nonaphatalene-8-sulphonic acid (ANS) fluorophore. The results show a d istinct influence of high pressure on the native dimers whose changes in conformation may, depending on whether or not these alterations are promptly reversed, be distinguished in elastic and inelastic changes. Elastic changes are ubiquitous and refer to pronounced modulations of the phosphorescence Lifetime which is a monitor of the internal flexi bility of the macromolecules. They attest to a tightening of the globu lar structure in the lower pressure range (below 1.5 kbar) as opposed to an increased fluidity in the higher range. The trend is similar bet ween the two proteins and the tightening/loosening effect is fully con sistent with the role that internal cavities and hydration of polypept ide is expected to play in determining the compressibility of these bi opolymers. Inelastic perturbations reveal a more profound loosening of the globular fold and were observed only with alcohol dehydrogenase u nder conditions (low temperature (t < 10 degrees C) and high pressure (p > 2.5 kbar)) that favour protein hydration. They involve slow conse cutive reactions that produce drastic reductions in phosphorescence li fetime, spectral red shifts, quenching of fluorescence and phosphoresc ence emission and modulation of ANS binding. Judging from the full pro tection afforded by glycerol as cosolvent, or the remarkable enhanceme nt caused by modest concentrations of urea, the driving force of these perturbations appears to be pressure-induced hydration of the protein . Inelastic conformational changes are accompanied by a slow and often incomplete recovery of enzymatic activity. The characteristic times o f these processes, their pressure dependence and the slow, thermally a ctivated, reversibility are discussed in the Light of hysteresis pheno mena and changes of subunit affinity in dissociation equilibria. (C) 1 996 Academic Press Limited