Fundamental molecular properties that promote the maintenance of heritable phenotypic variation and functional innovation are largely unknown. Structural constraints in a protein can hinder optimizing its 3D structure simultaneously for multiple functions if each function requires a different optimal structure. Promiscuous enzymes are prone to such constraints because their substrates for different functions may bind optimally to different structures of the enzyme. Any amino acid changes in the enzyme that improves the structure for one function may compromise another function. Such constraints can prevent the spreading of a beneficial amino acid variant and facilitate the origin of new genes and the diversification of protein families. Our lab uses molecular evolution theory, protein mutagenesis and functional assays, transgenics, and fitness assays to elucidate the roles that protein structural constraints play in maintaining phenotypic variation by balancing natural selection and the evolution of new genes and gene families.