Cavitands are effective templates for inducing stability and nativelike structure in de Novo four-helix bundles

We have designed, synthesized and characterized eight cavitand-based de nov o four-helix bundles, where each helix contains the basis sequence EELLKKLE ELLKKG. We find that each de novo protein is highly helical and extremely s table to the chemical denaturant guanidine hydrochloride (GuHCl). We studie d the effect of the cavitand-peptide linker on the stability of each de nov o protein. Flexible linkers render the helical structures more susceptible to denaturation by GuHCl. Linker structure and length also dictate monomer/ dimer equilibria of the proteins. Proteins containing 0-3 Cry units possess varying degrees of nativelike structure. In contrast, proteins containing two or four methylene Linkers are more characteristic of molten globules. T hese differences can be attributed to the additional hydrogen bonding capab ilities of the glycine linker variants and to the distance between the cavi tand template and the helical bundle. The cavitand pendent group was also c hanged in one de novo protein from a methyl group to a propyl-phosphate moi ety. This alteration does not affect the stability or packing within the he lical bundle; however, it does affect the monomer/dimer equilibrium.