Effect of multiple symmetries on the association of R67 DHFR subunits bearing interfacial complementing mutations

It was shown previously that complementation could be a powerful mean to probe protein–protein interactions in the normally tetrameric R67 DHFR. Indeed, mixing complementing inactive dimeric mutants produced active heterotetramers. This approach turned a homo‐oligomer into a hetero‐oligomer and thus allowed the use of combinatorial assays, a subtle analysis of the association forces, and a precise determination of the equilibrium dissociation constants ( K D ) by titrimetry. However, for some of the complementing pairs, the experimental data implied multiple equilibria involving heterodimers, although no monomers could be detected. Thus, the reactions involved had to be identified to elaborate a suitable model to determine the K D of those pairs correctly. That model suggested that homodimers associated rapidly before the protomers could be redistributed in a multiple equilibrium system. Kinetic data confirmed that view. The association data at equilibrium were analyzed by multiple curve fitting with all plausible combinations of parameters. This gave a confidence interval for K D that is safer than the usual 67% or 90% confidence interval. Finally, the K D of one specific reaction, the dissociation of a heterotetramer with the relevant symmetry into two homodimers could be determined with the relevant model for each complementing pair, although multiple equilibria were present. These K D can thus be used as a set of references data to test and improve theoretical methods such as association free energy calculations.