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Dissociation of lac repressor tetramer by high hydrostatic pressures was monitored with intrinsic tryptophan fluorescence. With the assumption of complete dissociation to monomer, tryptophan polarization data gave delta V a approximately 170 mL/mol and the concentration for 50% tetramer dissociation, C1/2, was 3.8 X 10(-8) M. Upon addition of inducer, the calculated delta V a increased to approximately 220 mL/mol and the C1/2 decreased to approximately 1 X 10(-8) M, a free energy difference of approximately 0.7 kcal. These results indicate a modest stabilization of the tetramer by the presence of inducer. Monitoring the average energy of tryptophan emission demonstrated that tetramer dissociation takes place over the same range of pressures as evidenced by the polarization data and IPTG dissociation can be more or less superimposed upon tetramer dissociation depending upon the ligand concentration used. Although the two transitions cannot be separated entirely, the delta V a for the region of the pressure dependence dominated by ligand dissociation was 69 mL/mol, an unexpectedly large value. For tetramer modified with methyl methanethiosulfonate, subunit dissociation was shifted to much higher pressures and IPTG dissociation did not occur. The delta V a for subunit association was calculated as approximately 160 mL/mol, and the C1/2 was 3.5 X 10(-9) M. Interactions at the subunit interface of the modified protein are apparently stronger than in the unmodified protein. The absence of inducer dissociation from the MMTS-modified tetramer by the application of high hydrostatic pressure suggests that the volume change for inducer binding to the modified protein is much smaller than that observed for the unmodified repressor.

Changes that occur in subunit neurophysin structure upon ligand binding were explored by two methods. First, the thermal coefficient of the viscosity around the subunit tyrosine was monitored, which yields information on the environmental flexibility and free rotational space of the fluorophore. Initially, it was determined that the environmental flexibility and the free space around each subunit tyrosine are unperturbed upon dimerization. Binding of the tripeptide analogue of oxytocin causes the once homologous environments of the subunit tyrosines to become drastically different such that one moves onto a closely packed environment whereas the other moves into a region of larger free space. Even though the subunits as seen by each tyrosine are very different, the specific binding sites as seen by the ligands are similar. It was also found that ligand binding is stabilized by ring stacking and that energy transfer occurs between the tyrosine of the ligand and the neurophysin subunit tyrosine. Second, changes in subunit structure upon ligation were also followed by the determination of the order of free energy coupling between ligand binding and oligomerization, which tells how each ligand affects the subunit affinity. Since the binding of ligand is cooperative and induces dimer formation, there is second-order coupling between ligand binding and dimerization and the binding of the second ligand is responsible for the increase in subunit affinity.