Figure 4.

Ratios of the predicted dissociation constant, K _D,pre, to the experimentally observed dissociation constant, K _D,ob for all the double mutants studied before (filled bars) and after (open bars) reduction by DTT. K _D,pre is estimated from the dissociation constants recorded for the single-cysteine mutant channels assuming independent binding of Cd²⁺ to the inserted cysteines in double-cysteine mutants (see methods, Eq. 1). For the mutants with ratios of K _D,ob/K _D,pre around 1, we assume that the two inserted cysteines are binding Cd²⁺ independently. Values of K _D,ob/K _D,pre statistically different from 1 are identified by asterisks (*). The mutant channels with K _D,ob/K _D,pre below 0.37 (i.e., with stabilization energies of 1 kT) are identified by open triangles.