HIGH HYDROSTATIC-PRESSURE INDUCES THE DISSOCIATION OF CPN60 TETRADECAMERS AND REVEALS A PLASTICITY OF THE MONOMERS

Hydrostatic pressures up to 2 kbar have been used to form monomers fro m the 14-subunit oligomer of the chaperonin, Cpn60. The fluorescence o f 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid (bisANS), followe d at high pressure, demonstrated an increase in hydrophobic exposure o n dissociation, Cpn60 dissociated with first order kinetics, The trans ition occurred between 1.3 and 2 kbar (P-50 = 1.75 kbar), and it was f acilitated by MgATP (P-50 = 1.1 kbar), With MgATP, the fluorescence sh owed a rapid first order phase (t1/2 = 3.7 min) in addition to a phase that was similar to the single phase for Cpn60 alone (t1/2 = 11.4 min ), The bisANS fluorescence decreased slowly after depressurization, an d the relaxation was faster at 25 degrees C (t1/2 = 58 h) than at 4 de grees C (t1/2 = 86 h) and faster still if the sample at 4 degrees C co ntained MgATP when it was pressurized (t1/2 = 18 h). There was no sign ificant effect if the MgATP was added after depressurization, Analytic al ultracentrifugation, after depressurization, confirmed that metasta ble monomers were produced that slowly reassociated to form the oligom ers (t1/2 = 150 h at 25 degrees C), Immediately after depressurization , the monomers (a) had all three sulfhydryl groups exposed for labelin g with 6-iodoacetamidofluorescein, (5) showed a proteolytic susceptibi lity that was intermediate between native Cpn60 and Cpn60 in 2.5 M ure a, and (c) were not able to capture a folding intermediate of the enzy me rhodanese, After incubation at atmospheric pressure, monomeric Cpn6 0 regained the ability to interact with rhodanese intermediates, and t he sulfhydryl reactivity fell before significantly reassociating to 14 -mers, The different rates of recovery of the native properties indica te that a complex series of conformational events occur following depr essurization. Finally, the monomers resulting from pressure were diffe rent from those produced from Cpn60 by the action of 2.5 M urea, These results demonstrate that there is a fast, pressure-induced dissociati on of the Cpn60 14-mer followed by a conformational drift of the disso ciated monomers that can be influenced by the presence of MgATP.