The helix-loop-helix (HLH) family of transcription factors regulate cellular growth and differentiation. The goal of this proposal is to understand on a molecular level the origins of binding affinity between HLH domains. These domains mediate protein-protein contacts among HLH transcription factors, altering their DNA-binding specificity and thus regulating transcription. Domains from the HLH transcription factors controlling myogenesis, MyoD, E47, and Id, will be used as a model system. The structures of the Id homo-oligomer as well as the hetero-oligomers will be studied crystallographically, their binding energies will be measured, and hypotheses for their relative binding affinities will be tested through mutagenesis. From these results, we will generate an algorithm to predict the pairing of HLH sequences. Developing a set of rules for HLH domain interactions will enable us to design specific probes for the expression of transcription factors in vivo. Ultimately this information may form the basis for new strategies to restore the normal function of HLH transcription factors that cause uncontrolled proliferation and de-differentiation in tumors.