Heme proteins serve a wide variety of functions in the biological milieu.1 Our approach to the study of protein structure function relationships is to use insight drawn from nature to design and synthesize minimal peptides which fold, assemble, and incorporate cofactors.2-8 Molecular maquettes2 are functional synthetic proteins which are simplified versions of their biological counterparts. This strategy is aimed to provide direct insight into protein design,9-18 cofactor incorporation,19-21 structure and function properties of redox proteins, and establishment of in situ catalytic properties. The four R-helix bundle22,23 has been successfully used in this manner for construction of maquettes containing hemes,2,7 porphyrin dimers,3 flavins,4 iron sulfur clusters,5 and amino acid radicals.6 Several of the heme-containing maquettes have been analyzed by NMR spectroscopy and show properties atypical of native-like structure. This Communication describes recent progress toward the design and structural characterization of a native-like maquette scaffold. Like the majority of designed proteins24 the prototype heme protein maquette,2 H10H24 (Ac-CGGGELWKL‚HEELLKK‚FEELLKL‚HEERLKK‚L-CONH2), shows characteristics associated with non-native protein structure. We have explored computationally and experimentally the effect of substituting leucine residues with conformationally restricted â-branched and/or aromatic amino acids to increase hydrophobic core packing specificity. Since the three internal heptad a positions25 (H10, F17, and H24) of H10H24 are engineered for heme binding and R27 is designed to modulate the heme redox potential, the best candidates for redesign were the leucine residues L6, L13, and L20 (see Figure 1A). Initial studies have focused on exchanging L6 for isoleucine and L13 for phenylalanine. Single and double variants at these two helix positions (H10H24-L6I, H10H24-L13F, and H10H24-L6I,L13F) were prepared,26 and the effect of these substitutions on the coiledcoil thermodynamic stability and structural specificity determined. This series of maquettes displays a continuum of changes ranging from the poorly ordered and least stable prototype H10H24 to the uniquely structured and most stable double variant H10H24-L6I,L13F.