Controlling topology and native-like behavior of de novo-designed peptides: design and characterization of antiparallel four-stranded coiled coils.

The de novo design of peptides and proteins has emerged as an attractive approach for investigating protein structure and function. Here, the design, synthesis, and characterization of a new series of alpha-helical peptides intended to form antiparallel four-stranded coiled coils is described. Computer models were generated without the use of extant protein structures and were used to refine the sequence. The peptides are of the general formula Ncap-(XaZbZcLdZeZfZg)3-Ccap, where X is either Ala, Val, Thr, or Leu, and Ncap and Ccap are sequences designed to satisfy the helices unpaired amide nitrogens and carbonyl oxygens, respectively. The hydrophobic residues (at positions a and d) were chosen so that geometric packing of large and small hydrophobes would favor an antiparallel arrangement. Special attention was also given to residues at the helix--helix interfaces. These residues were chosen to balance potential attractive and repulsive electrostatic forces so that the desired topology was favored while other possible folds were destabilized. Two of the four peptides associate under neutral conditions into the desired tetramers. One of the complexes (a = Val) behaves like a native-like protein as judged by NMR, thermodynamics, and apolar dye (ANS) binding. The other tetrameric complex (a = Leu) exhibits broader NMR resonances, diminished values of delta H and delta Cp, and tight binding of the hydrophobic dye ANS, similar to early designed proteins. These results reinforce the importance of optimizing van der Waals packing interactions in protein design but demonstrate that hydrophobic packing must be balanced with hydrogen-bonding and electrostatic interactions to produce novel native-like proteins.