MYOD-E12 HETERODIMERS AND MYOD-MYOD HOMODIMERS ARE EQUALLY STABLE

Muscle development is controlled by the MyoD family of basic helix-loo p-helix (bHLH) DNA-binding proteins, These proteins dimerize with ubiq uitous products of the E2A gene (E12 and E47) and bind in a sequence-s pecific manner to enhancer regions of muscle-specific genes activating their expression. In this study, fluorescence anisotropy has been uti lized to characterize the interactions of recombinant MyoD and E12 in solution in the absence of DNA. The Gibb's free energies of dissociati on (Delta G) and the equilibrium dissociation constants (K-D) for the protein-protein interactions are reported, The Delta G for the MyoD ho modimers in 100 mM KCl was 8.7 kcal/mol (K-D = 340 nM), and increasing the salt concentration resulted in destabilization of the dimer. From titrations of MyoD-dansyl with E12 at 100 mM KCl, a free energy of he terodimerization of 8.7 (+0.4/-2.4) kcal/mol was recovered using rigor ous confidence limit testing. The titrations of E12-dansyl with MyoD y ielded a free energy of 8.3 kcal/mol with tighter confidence limits, 0.5/-0.8 kcal/mol. Thus, in the absence of DNA, both MyoD homodimers a nd MyoD-E12 heterodimers are relatively weak complexes of approximatel y the same stability, E12 does not form stable homo-oligomeric complex es; remaining monomeric at concentrations as high as 20 mu M. Based on these results and the apparent binding constants reported previously for DNA binding, DNA is likely to facilitate the dimerization of MyoD and E12. Furthermore, higher affinity interactions of MyoD-E12 heterod imers versus MyoD homodimers with DNA binding sites is not due to pref erential heterodimerization.