| Abstract
| - Muscle development is controlled by the MyoD family ofbasic helix−loop−helix (bHLH)DNA-binding proteins. These proteins dimerize with ubiquitousproducts of the E2A gene (E12 andE47) and bind in a sequence-specific manner to enhancer regions ofmuscle-specific genes activatingtheir expression. In this study, fluorescence anisotropy has beenutilized to characterize the interactionsof recombinant MyoD and E12 in solution in the absence of DNA. TheGibb's free energies of dissociation(ΔG) and the equilibrium dissociation constants(KD) for the protein−protein interactions arereported.The ΔG for the MyoD homodimers in 100 mM KCl was 8.7kcal/mol (KD = 340 nM), and increasingthesalt concentration resulted in destabilization of the dimer. Fromtitrations of MyoD−dansyl with E12 at100 mM KCl, a free energy of heterodimerization of 8.7 (+0.4/−2.4)kcal/mol was recovered using rigorousconfidence limit testing. The titrations of E12-dansyl with MyoDyielded a free energy of 8.3 kcal/molwith tighter confidence limits, +0.5/−0.8 kcal/mol. Thus, inthe absence of DNA, both MyoD homodimersand MyoD−E12 heterodimers are relatively weak complexes ofapproximately the same stability. E12does not form stable homo-oligomeric complexes; remaining monomeric atconcentrations as high as 20μM. Based on these results and the apparent binding constantsreported previously for DNA binding,DNA is likely to facilitate the dimerization of MyoD and E12.Furthermore, higher affinity interactionsof MyoD−E12 heterodimers versus MyoD homodimers with DNA bindingsites is not due to preferentialheterodimerization.
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