DE-NOVO DESIGN OF NATIVE PROTEINS - CHARACTERIZATION OF PROTEINS INTENDED TO FOLD INTO ANTIPARALLEL, ROP-LIKE, 4-HELIX BUNDLES

The de novo design and characterization of a series of 51-residue heli x-turn-helix peptides intended to dimerize into antiparallel four-stra nded coiled coils is described. The sequence is based on a coiled coil heptad repeat urn-(X(a)Z(b)Z(c)L(d)Z(e)Z(f)Z(g))(3)-C-cap-CONH2, wher e X is either Val or Ala. The overall topology was intended to be simi lar to that found in the Escherichia coli protein ROP. The design stra tegy included consideration of geometric complementarity of the packin g of side chains within the hydrophobic core as well as the use of spe cific interfacial interactions, both of which were intended to favor t he desired POP-like topology. Additionally, the sequence was designed to destabilize potential alternative structures that might compete wit h the desired topology. The peptides (RLP-1, RLP-2, and RLP-3) assembl e into stable alpha-helical dimers and exhibit the hallmarks of a nati ve protein as judged by its spectroscopic properties, and the lack of binding to hydrophobic dyes. Also, the enthalpy and heat capacity chan ges upon denaturation were determined by measuring the temperature dep endence of the CD spectra and confirmed by differential scanning calor imetry (DSC). The values determined by the two methods are in excellen t agreement and are in the range of these of naturally occurring prote ins of this size. These results suggest that it is now possible to des ign native-like helical proteins that should serve as templates for th e further design of functional proteins.