Modulation of the stability of a gene-regulatory protein dimer by DNA and cAMP.

We describe an experimental approach to the measurement of protein subunit exchange in which biotinylated subunits mediate attachment of 35S-labeled subunits to a streptavidin column as a result of the exchange process. Application of the method to Escherichia coli catabolite activator protein (CAP) revealed that in the absence of cAMP, the dimerization equilibrium constant is 3 x 10(10) M-1, with a dimer lifetime of 300 min. Exchange of CAP subunits is accelerated at least 1000-fold by the presence of nonspecific DNA, under low ionic strength conditions. Catalysis of exchange also occurs at physiological ionic conditions. In contrast, physiological concentrations of cAMP stabilize CAP with respect to subunit exchange in either the presence or the absence of DNA. We discuss the functional implications of monomerization of gene-regulatory proteins resulting from kinetic and thermodynamic lability of their dimers.