Solution structure of a de novo helical protein by 2D-NMR spectroscopy.

The de novo design of proteins represents a critical test for our knowledge of protein structure. However, no structure of a successfully designed protein has yet been reported. This paper reports the design and the tertiary structure of a de novo 35-residue helix-loop-helix protein (ALIN), with helices arranged in an anti-parallel manner. For the helical region, we designed sequences that have high helicities even as isolated peptides. A flexible loop containing glycine residues was used to connect the two helices and the interhelical hydrophobic interaction was stabilized by the introduction of an SS-bridge. This arrangement of two consecutive helices bridged by a disulfide bond is not observed in any natural proteins. The protein is highly soluble and monomeric in aqueous solution. The structure of ALIN, determined by 1H-NMR and distance geometry calculation, agrees well with the design. In addition, the exchange rates of amide protons indicate the presence of stable hydrogen bonds in the helical region. ALIN is the first de novo designed protein with a stable tertiary fold confirmed by NMR.