High hydrostatic pressure induces the dissociation of cpn60 tetradecamers and reveals a plasticity of the monomers

Abstract

Hydrostatic pressures up to 2 kbar have been used to form monomers from the 14-subunit oligomer of the chaperonin, Cpn60. The fluorescence of 1,1'- bi(4-anilino)naphthalene-5,5'-disulfonic acid (bisANS), followed at high pressure, demonstrated an increase in hydrophobic exposure on dissociation. Cpn60 dissociated with first order kinetics. The transition occurred between 1.3 and 2 kbar (P50 = 1.75 kbar), and it was facilitated by MgATP (P50 = 1.1 kbar). With MgATP, the fluorescence showed a rapid first order phase (t 1/2 = 3.7 min) in addition to a phase that was similar to the single phase for Cpn60 alone (t 1/2 = 11.4 min). The bisANS fluorescence decreased slowly after depressurization, and the relaxation was faster at 25 °C (t 1/2 = 58 h) than at 4 °C (t 1/2 = 86 h) and faster still if the sample at 4 °C contained MgATP when it was pressurized (t 1/2 = 18 h). There was no significant effect if the MgATP was added after depressurization. Analytical ultracentrifugation, after depressurization, confirmed that metastable monomers were produced that slowly reassociated to form the oligomers (t 1/2 = 150 h at 25 °C). Immediately after depressurization, the monomers (a) had all three sulfhydryl groups exposed for labeling with 6-iodoacetamidofluorescein, (b) showed a proteolytic susceptibility that was intermediate between native Cpn60 and Cpn60 in 2.5 M urea, and (c) were not able to capture a folding intermediate of the enzyme rhodanese. After incubation at atmospheric pressure, monomeric Cpn60 regained the ability to interact with rhodanese intermediates, and the sulfhydryl reactivity fell before significantly reassociating to 14-mers. The different rates of recovery of the native properties indicate that a complex series of conformational events occur following depressurization. Finally, the monomers resulting from pressure were different from those produced from Cpn60 by the action of 2.5 M urea. These results demonstrate that there is a fast, pressure-induced dissociation of the Cpn60 14-mer followed by a conformational drift of the dissociated monomers that can be influenced by the presence of MgATP.