Design of λ Cro Fold: Solution Structure of a Monomeric Variant of the De Novo Protein

One of the classical DNA-binding proteins, bacteriophage λ Cro, forms a homodimer with a unique fold of α-helices and β-sheets. We have computationally designed an artificial sequence of 60 amino acid residues to stabilize the backbone tertiary structure of the λ Cro dimer by simulated annealing using knowledge-based structure-sequence compatibility functions. The designed amino acid sequence has 25% identity with that of natural λ Cro and preserves Phe58, which is important for formation of the stably folded structure of λ Cro. The designed dimer protein and its monomeric variant, which was redesigned by the insertion of a β-hairpin sequence at the C-terminal region to prevent dimerization, were synthesized and biochemically characterized to be well folded. The designed protein was monomeric under a wide range of protein concentrations and its solution structure was determined by NMR spectroscopy. The solved structure is similar to that of a monomeric variant of natural λ Cro with a root-mean-square deviation of the polypeptide backbones at 2.1 Å and has a well-packed protein core. Thus, our knowledge-based functions provide approximate but essential relationships between amino acid sequences and protein structures, and are useful for finding novel sequences that are foldable into a given target structure.