Dissociation of a native dimer to a molten globule monomer. Effects of pressure and dilution on the association equilibrium of arc repressor.

The monomer-dimer association reaction of Arc repressor was studied by pressure-induced dissociation and by dilution. The dissociation was measured by the decrease (red shift) in the average energy of emission of the tryptophan fluorescence. Pressure dissociation also promoted a decrease in the excited-state lifetime of the single tryptophanyl residue, Trp14. These observations suggest that Trp14 becomes exposed to an aqueous environment following dissociation. The pressure-dissociation curves were concentration dependent, with p1/2 (half-dissociation pressure) shifting to higher pressures as the concentration increased. The dissociation constant (KdO) obtained by extrapolating the pressure-dissociation curves to atmospheric pressure was similar to that determined from the dilution curve (KdO = 30 nM). An anomalous steepness of dissociation in response to dilution was observed, suggesting that conformational changes occur as a result of dissociation of Arc repressor. Binding of bis(8-anilinonaphthalene-1-sulfonate) to Arc repressor was not significantly affected by pressure dissociation, whereas thermal or urea denaturation was accompanied by a dramatic decrease in binding. These results suggest that the conformational changes that follow dissociation induced by pressure are more limited than those following denaturation. The tryptophan anisotropy decreased by about one-half, suggesting the dissociation of a globular dimer to a compact monomer. On the other hand, denaturation by urea promoted an increase in anisotropy, as expected for a random-coil conformation. Dissociated Arc has the hydrodynamic properties of a folded monomer. On the other hand, dissociated Arc has a high degree of exposure of hydrophobic side-chains, and the distribution of conformations is much broader than that in the folded dimer. These features suggest that the dissociated subunit is a molten globule. The subunit interaction was substantially increased by a single amino acid substitution (Pro8----Leu), and the free energy of stabilization amounted to -2.9 kcal/mol. This increased stability suggests that residue 8 is located in the dimer interface and that part of the tertiary and most of the quaternary structure constraints result from the interaction between the intersubunit beta-strands.